Nitrate-Dependent Control of Shoot K Homeostasis by the Nitrate Transporter1/Peptide Transporter Family Member NPF7.3/NRT1.5 and the Stelar K+ Outward Rectifier SKOR in Arabidopsis

Root-to-shoot translocation and shoot homeostasis of potassium (K) determine nutrient balance, growth, and stress tolerance of vascular plants. To maintain the cation-anion balance, xylem loading of K⁺ in the roots relies on the concomitant loading of counteranions, like nitrate (NO₃⁻). However, the coregulation of these loading steps is unclear. Here, we show that the bidirectional, low-affinity Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) family member NPF7.3/NRT1.5 is important for the NO₃⁻-dependent K⁺ translocation in Arabidopsis (Arabidopsis thaliana). Lack of NPF7.3/NRT1.5 resulted in K deficiency in shoots under low NO₃⁻ nutrition, whereas the root elemental composition was unchanged. Gene expression data corroborated K deficiency in the nrt1.5-5 shoot, whereas the root responded with a differential expression of genes involved in cation-anion balance. A grafting experiment confirmed that the presence of NPF7.3/NRT1.5 in the root is a prerequisite for proper root-to-shoot translocation of K⁺ under low NO₃⁻ supply. Because the depolarization-activated Stelar K⁺ Outward Rectifier (SKOR) has previously been described as a major contributor for root-to-shoot translocation of K⁺ in Arabidopsis, we addressed the hypothesis that NPF7.3/NRT1.5-mediated NO₃⁻ translocation might affect xylem loading and root-to-shoot K⁺ translocation through SKOR. Indeed, growth of nrt1.5-5 and skor-2 single and double mutants under different K/NO₃⁻ regimes revealed that both proteins contribute to K⁺ translocation from root to shoot. SKOR activity dominates under high NO₃⁻ and low K⁺ supply, whereas NPF7.3/NRT1.5 is required under low NO₃⁻ availability. This study unravels nutritional conditions as a critical factor for the joint activity of SKOR and NPF7.3/NRT1.5 for shoot K homeostasis.The macronutrient potassium (K) is essential for plant growth and development because of its crucial roles in various cellular processes (i.e. regulation of enzyme activities), stabilization of protein synthesis, and neutralization of negative charges. In addition, it is a major component of the cation-anion balance and osmoregulation in plants, thereby influencing cellular turgor, xylem and phloem transport, pH homeostasis, and the setting of membrane potentials (Maathuis, 2009; Marschner, 2012; Sharma et al., 2013). K⁺ uptake and distribution in Arabidopsis (Arabidopsis thaliana) are accomplished by a total of 71 membrane proteins that have been assigned to five gene families: the Shaker and Tandem-Pore K⁺ channels (now also including the inward-rectifier K-like (K_ir-like) channels), the K⁺ uptake permeases (KUP/HAK/KT), the K⁺ transporter (HKT) family, and the cation proton antiporters (CPA; Gierth and Mäser, 2007; Gomez-Porras et al., 2012; Sharma et al., 2013).Root xylem loading is a key step for the delivery of nutrients to the shoot (Poirier et al., 1991; Engels and Marschner, 1992a; Gaymard et al., 1998; Takano et al., 2002; Park et al., 2008). Root-to-shoot translocation of K⁺ is mediated by the voltage-dependent Shaker family K⁺ channel Stelar K⁺ Outward Rectifier (SKOR). The gene is primarily expressed in pericycle and root xylem parenchyma cells, and it is down-regulated upon K shortage and in response to treatments with the phytohormones abscisic acid, cytokinin, and auxin. Such gene expression changes are thought to control K⁺ secretion into the xylem sap and K⁺ reallocation through the phloem to adjust root K⁺ transport activity to K⁺ availability and shoot demand (Pilot et al., 2003). SKOR is activated upon membrane depolarization, and it is in a closed state when the driving force for K⁺ is inwardly directed. It elicits outward K⁺ currents, facilitating the release of the cation from the cells into the xylem. The voltage dependency of the channel is modulated by the external K⁺ concentration to minimize the risk of an undesired K⁺ influx under high K⁺ availability (Johansson et al., 2006). Root-to-shoot K⁺ transfer was strongly reduced in the knockout mutant skor-1, resulting in a decreased shoot K content, whereas the root K content remained unaffected (Gaymard et al., 1998).Root xylem loading is subject to the maintenance of a cation-anion balance, and nitrate (NO₃⁻) is the quantitatively most important anion counterbalancing xylem loading of K⁺ (Engels and Marschner, 1993). Members of the Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) transporter family (NPF) play a prominent role in NO₃⁻ uptake and allocation in Arabidopsis (summarized in Krouk et al., 2010; Wang et al., 2012; and Léran et al., 2014). Two of them have recently been reported to control xylem NO₃⁻ loading and unloading. The low-affinity, pH-dependent bidirectional NO₃⁻ transporter NPF7.3/NRT1.5 (subsequently termed NRT1.5) mediates NO₃⁻ efflux from pericycle cells to the xylem vessels, whereas the low-affinity influx protein NPF7.2/NRT1.8 removes NO₃⁻ from the xylem sap and transfers it into xylem parenchyma cells (Lin et al., 2008; Li et al., 2010; Chen et al., 2012). Accordingly, the expression of both genes is oppositely regulated under various stress conditions (Li et al., 2010). In nrt1.5 mutants, NRT1.8 expression is increased, which is thought to enhance NO₃⁻ reallocation to the root (Chen et al., 2012).The NRT1.5 gene is mainly expressed in root pericycle cells close to the xylem, and the protein localizes to the plasma membrane. In nrt1.5 mutants, less NO₃⁻ is transported from the root to the shoot, and the NO₃⁻ concentration in the xylem sap is reduced. However, root-to-shoot NO₃⁻ transport is not completely abolished in these mutants, indicating the existence of additional xylem-loading activities for NO₃⁻ (Lin et al., 2008; Wang et al., 2012). The recent observation that NPF6.3/NRT1.1/CHL1 and NPF6.2/NRT1.4 are also capable of mediating bidirectional NO₃⁻ transport in Xenopus laevis oocytes might indicate that more NPF family members are contributing to xylem loading with NO₃⁻ (Léran et al., 2013).Electrophysiological studies with NRT1.5-expressing X. laevis oocytes revealed that NO₃⁻ excited an inward current at pH 5.5, which would be expected for a proton-coupled nitrate transporter with a proton to nitrate ratio larger than one (Lin et al., 2008). The inward currents elicited by exposure to nitrate were pH dependent, and Lin et al. (2008) observed that NRT1.5 can also facilitate nitrate efflux when the oocytes were incubated at pH 7.4. Lin et al. (2008) concluded that NRT1.5 can transport nitrate in both directions, presumably through a proton-coupled mechanism. Interestingly, a K⁺ gradient was not sufficient to drive NRT1.5-mediated NO₃⁻ export. However, the determination of root and shoot cation concentrations in the nrt1.5-1 mutant revealed that the amount of K⁺ translocated to the shoot was reduced when NO₃⁻ but not NH₄⁺ was supplied as the N source. Therefore, Lin et al. (2008) suggested a regulatory loop between NO₃⁻ and K⁺ at the xylem loading step.A close relationship between these two nutrients concerning uptake, translocation, recycling, and reduction (of NO₃⁻) has been described in physiological studies since the 1960s (e.g. Ben Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985), but only recently, common components in the NO₃⁻ and K⁺ uptake pathways were identified and led to the first ideas of how such a cross talk might be coordinated on the molecular level. The uptake activity of the K⁺ channel AKT1 as well as the affinity of the NO₃⁻ transporter NPF6.3/NRT1.1/CHL1 are both modulated by the activity of CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE23 (CIPK23), which itself is regulated by CALCINEURIN B-LIKE PROTEIN9 (CBL9) under both deficiencies (Xu et al., 2006; Ho et al., 2009). Yet, the details of this interaction in root K⁺ uptake, the (regulation of) xylem loading with K⁺ and NO₃⁻, and the involvement of SKOR and NRT1.5 in this process are unknown.In this study, we approached this problem by investigating the molecular and physiological responses of Arabidopsis wild-type (Columbia-0 [Col-0]), nrt1.5, and skor transfer DNA (T-DNA) insertion lines to varying NO₃⁻ and K⁺ regimes. The nrt1.5 mutant developed an early senescence phenotype under low NO₃⁻ nutrition, which could be attributed to a reduced K⁺ translocation to the shoot. The assessment of nrt1.5 and skor single- and double-knockout lines disclosed an interplay of the two proteins in the NO₃⁻-dependent control of shoot K homeostasis. The presented data indicate that SKOR mediates K⁺ root-to-shoot translocation under high NO₃⁻ and low K⁺ availability, whereas NRT1.5 is important for K⁺ translocation under low NO₃⁻ availability, irrespective of the K⁺ supply.     

The K+ and NO3− Interaction Mediated by NITRATE TRANSPORTER1.1 Ensures Better Plant Growth under K+-Limiting Conditions

K⁺ and NO₃⁻ are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO₃⁻ and K⁺ in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K⁺ uptake and root-to-shoot allocation, and were characterized by growth arrest under K⁺-limiting conditions. The K⁺ uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K⁺ contents in nrt1.1-1/K⁺ transporter1, nrt1.1-1/high-affinity K⁺ transporter5-3, nrt1.1-1/K⁺ uptake permease7, and nrt1.1-1/stelar K⁺ outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K⁺ channels/transporters located in the root epidermis–cortex and central vasculature. Further study revealed that these K⁺ uptake-related interactions are dependent on an H⁺-consuming mechanism associated with the H⁺/NO₃⁻ symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis–cortex and central vasculature are coordinated with K⁺ channels/transporters to improve K⁺ uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K⁺-limiting conditions.

Potassium (K) is an essential element for plant growth and development and contributes to determining the yield and quality of crops in agriculture production (Wang and Wu, 2013). However, the concentrations of soluble K⁺ in most soils are relatively low, which often limits plant growth (Maathuis, 2009). Although crop production can be increased by applying large amounts of potassic fertilizers to agricultural fields, only approximately one-half of the applied fertilizers is available to plants; the remainder accumulates as residues in soils, consequently leading to environmental contamination (Meena et al., 2016). Therefore, there is a pressing need to gain a more complete understanding of the molecular mechanisms underlying K⁺ transport and regulation in order to enhance the K⁺ utilization efficiency of plants. Accordingly, in the past few decades, researchers have focused on identifying K⁺ channels and transporters in plants, as well as the mechanisms underlying their regulation.In Arabidopsis (Arabidopsis thaliana), 71 K⁺ channels and transporters have been identified and categorized into three channel (Shaker, Tandem-Pore K⁺, and K_ir-like) and three transporter (K⁺ uptake permeases [KT/HAK/KUP], High-affinity K⁺ transporters [HKT], and cation/proton antiporter [CPA]) families (Wang and Wu, 2010). Among these, the shaker inward K⁺ channel K⁺ TRANSPORTER1 (AKT1) and the KT/HAK/KUP K⁺ transporter HIGH-AFFINITY K⁺ TRANSPORTER5 (HAK5) have been characterized as the two major components that contribute to K⁺ uptake in roots, although they have been found to operate at different K⁺ levels (Pyo et al., 2010; Wang and Wu, 2013). AKT1 functions in plant K⁺ uptake over a wide range of K⁺ concentrations, whereas HAK5 shows high-affinity K⁺ transport activity (Gierth et al., 2005). Following its uptake into root epidermal cells, K⁺ is distributed to different plant organs or tissues. The Arabidopsis shaker-like outward-rectifying K⁺ channel STELAR K⁺ OUTWARD RECTIFIER (SKOR), the expression of which was first identified in stelar tissues, has been shown to facilitate K⁺ secretion into xylem sap, which is a critical step in long-distance K⁺ transport from roots to shoots (Gaymard et al., 1998). Recently, K⁺ UPTAKE PERMEASE7 (KUP7), a member of the KT/HAK/KUP family, was functionally characterized as a K⁺ transporter participating in both root K⁺ uptake and root-to-shoot K⁺ allocation, particularly under K⁺-limiting conditions (Han et al., 2016). However, the uptake affinity for K⁺ has been found to be considerably lower in KUP7 than in HAK5 (Wang and Wu, 2017).In addition to the aforementioned K⁺ channels and transporters, other mineral elements, including Na⁺, Ca²⁺, and N, are known to have pronounced effects on K⁺ nutrition in plants. Given that N is the nutrient that is required in the greatest quantity by most plants and is the most widely used fertilizer nutrient in crop production, the relationships between N and K have long been investigated (Fageria and Baligar, 2005; Wang and Wu, 2013; Meng et al., 2016; Shin, 2017). Since the 1960s, physiological studies have revealed a close relationship between NO₃⁻ and K⁺ with regard to uptake and translocation (Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985; Drechsler et al., 2015). However, the coordination between these two nutrients in plant transport pathways remains to be extensively studied at the molecular level. We hypothesized that transporters involved in the transference of NO₃⁻ across cell membranes may play a role in controlling K⁺ nutrition in plants. Recently, NITRATE TRANSPORTER1.5 (NRT1.5), a member of the nitrate transporter1/peptide transporter family (NPF), initially identified as a pH-dependent bidirectional NO₃⁻ transporter (Lin et al., 2008), was shown to be involved in the control of K⁺ allocation in plants (Drechsler et al., 2015; Li et al., 2017; Du et al., 2019). Nevertheless, it was subsequently established that this function was merely associated with its role as a proton-coupled H⁺/K⁺ antiporter for K⁺ loading into the xylem (Li et al., 2017; Du et al., 2019), which is not associated with the transport of NO₃⁻. In this study, we showed that the loss of another nitrate transporter1 member, NRT1.1/NPF6.3, in nrt1.1 mutants led to the development of a more pronounced K⁺-deficiency phenotype under conditions of low-K⁺ stress. Further physiological and genetic evidence revealed that both the uptake and root-to-shoot allocation of K⁺ in plants require NRT1.1. However, NRT1.1 acts as a coordinator rather than a K⁺ channel/transporter in K⁺ uptake and root-to-shoot allocation, which could depend on its NO₃⁻-related transport activity. Our findings highlight the significance of nutrients and nutrient interactions in ensuring plant growth, and indicate that the modification of NRT1.1 homolog activity in crops using biological engineering techniques might be a promising approach that could simultaneously contribute to enhancing the utilization efficiencies of K and N fertilizers in agricultural production.     

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.Cadmium is highly toxic to humans (Nicholson et al., 1983), and its primary route of entry into the body is through crops grown in cadmium-contaminated soil (Clemens et al., 2013). A recent survey indicated that vegetables and rice (Oryza sativa) account for approximately 40% and 38%, respectively, of total cadmium exposure in residents of Shanghai, China’s largest city (He et al., 2013). However, cadmium contamination of agricultural soils as a result of rapid industrial development and release of agrochemicals into the environment is an increasingly serious problem. Many strategies have been proposed for remediating cadmium-contaminated soil to prevent cadmium uptake by crops. These strategies include the dig-and-dump method or encapsulation of the contaminated soil, chemical immobilization or extraction of cadmium, and phytoremediation by cadmium-hyperaccumulating plants (Pulford and Watson, 2003). However, the dig-and-dump and chemical methods are expensive, whereas phytoremediation requires several growing seasons to be effective, making it impractical in regions where farmland is limited and food supply insufficient.The shortfalls of these strategies have prompted researchers to develop alternative techniques that are cost-effective and interfere less with crop production. Use of nitrogen fertilizers is one of the most important agronomic practices and it has been suggested that their appropriate use might provide a relatively inexpensive, time-saving, and effective strategy for reducing cadmium entry into, and accumulation in, crops because NO₃⁻ facilitates cadmium uptake in hydroponically grown plants (Sarwar et al., 2010; Luo et al., 2012). However, in a preliminary study, we found that, in plants grown in soil, the effect of the nitrogen form on cadmium accumulation was strongly associated with the pH-buffering capacity of the soil. In soil with a lower pH-buffering capacity, application of ammonium (NH₄⁺) resulted in higher cadmium levels in plants than application of NO₃⁻, probably as a result of soil acidification by NH₄⁺, and the opposite effect was seen when plants were grown in soil with higher pH-buffering capacity (S.K. Fan, S.T. Du, and C.W. Jin, unpublished data). This suggests that management of the use of nitrogen fertilizers to prevent cadmium entry into crops might be difficult because of the wide variation in soil pH-buffering capacity.Because NO₃⁻ facilitates cadmium uptake in hydroponically grown plants as described above, modification of NO₃⁻ uptake pathways in plants might also affect cadmium uptake, in which case modifying these pathways to reduce cadmium entry into crops could circumvent the risks and the difficulties involved in nitrogen fertilizer management. Exposure to cadmium has been shown to reduce NO₃⁻ uptake by roots (Hernández et al., 1997; Gouia et al., 2000; Rizzardo et al., 2012), but this has been assumed to be deleterious to plant growth (Finkemeier et al., 2003; Rizzardo et al., 2012). The process by which NO₃⁻ is taken up across the root plasma membrane is complex, and several nitrate transporters (NRTs) involved in NO₃⁻ uptake from the growth medium have been characterized. In Arabidopsis (Arabidopsis thaliana), NRT1.1 is a dual-affinity transporter involved in both high- and low-affinity uptake, NRT1.2 is involved only in low-affinity NO₃⁻ uptake, whereas NRT2.1, NRT2.2, and NRT2.4 are only involved in high-affinity NO₃⁻ uptake (Wang et al., 2012; Léran et al., 2014). However, the transporter responsible for the cadmium-induced decrease in NO₃⁻ uptake remains unknown. Given the presumed association between NO₃⁻ uptake and cadmium uptake, it is important to identify the molecular mechanism involved in this process, and it is particularly important to determine whether the modulation of relevant NO₃⁻ transporters affects cadmium entry into plants.In this study, we investigated the relationship between NO₃⁻ uptake and cadmium uptake in Arabidopsis roots. To our knowledge, our results reveal a new mechanism for resisting cadmium toxicity: Cadmium reduces NO₃⁻ uptake by inhibiting NRT1.1 activity, which in turn reduces cadmium entry into root cells. As a result, cadmium levels in plants are lower and plant growth is improved. Our findings may provide a strategy for minimizing cadmium accumulation in crops grown in contaminated soil using biotechnological pathways to decrease NO₃⁻ uptake.     

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and β-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

Nitrate transporter/transceptor NRT1.13 monitors xylem 12 nitrate level to regulate shoot architecture and flowering time.     

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.     

The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg²⁺ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA2+, while MgA2+ together with Arg233 stabilize the inorganic phosphate (P_i); (ii) Asp168 and three water molecules coordinate MgB2+, and MgB2+ further stabilizes P_i by forming a hydrogen bond with two oxygens of P_i; (iii) Arg201 directly coordinates the P_i; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA2+, MgB2+ and P_i. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP⁺/NAD⁺) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.     

NRT1.1B improves selenium concentrations in rice grains by facilitating selenomethinone translocation

Selenium (Se) is an essential trace element for humans and other animals, yet approximately one billion people worldwide suffer from Se deficiency. Rice is a staple food for over half of the world's population that is a major dietary source of Se. In paddy soils, rice roots mainly take up selenite. Se speciation analysis indicated that most of the selenite absorbed by rice is predominantly transformed into selenomethinone (SeMet) and retained in roots. However, the mechanism by which SeMet is transported in plants remains largely unknown. In this study, SeMet uptake was found to be an energy‐dependent symport process involving H⁺ transport, with neutral amino acids strongly inhibiting SeMet uptake. We further revealed that NRT1.1B, a member of rice peptide transporter (PTR) family which plays an important role in nitrate uptake and transport in rice, displays SeMet transport activity in yeast and Xenopus oocyte. The uptake rate of SeMet in the roots and its accumulation rate in the shoots of nrt1.1b mutant were significantly repressed. Conversely, the overexpression of NRT1.1B in rice significantly promoted SeMet translocation from roots to shoots, resulting in increased Se concentrations in shoots and rice grains. With vascular‐specific expression of NRT1.1B, the grain Se concentration was 1.83‐fold higher than that of wild type. These results strongly demonstrate that NRT1.1B holds great potential for the improvement of Se concentrations in grains by facilitating SeMet translocation, and the findings provide novel insight into breeding of Se‐enriched rice varieties.