Molybdate Transport Research Articles

Regulation of Oryza sativa molybdate transporter1;3 degradation via RING finger E3 ligase OsAIR3

High concentrations of As in contaminated environments pose a serious threat to plant, human, and animal health. In this study, we characterized an As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene under arsenate (AsV) stress, named as Oryza sativa As-Induced RING E3 ligase 3 (OsAIR3). AsV treatment highly induced the expression of OsAIR3. OsAIR3-EYFP was localized to the nucleus in rice protoplasts and exhibited E3 ligase activity. Yeast two-hybrid screening and bimolecular fluorescence complementation and pull-down assays revealed the interaction of OsAIR3 with an O. sativa molybdate transporter (OsMOT1;3) in the plasma membrane and cytoplasm. In addition, an in vitro cell-free degradation assay was performed to demonstrate the degradation of OsMOT1;3 by OsAIR3 via the 26S proteasome system. Heterogeneous overexpression of OsAIR3 in Arabidopsis yielded AsV-tolerant phenotypes, as indicated by the comparison of cotyledon expansion, root elongation, shoot fresh weight, and As accumulation between the OsAIR3-overexpressing and control plants. Collectively, these findings suggest that OsAIR3 positively regulates plant response to AsV stress.

Marinobacter atlanticus electrode biofilms differentially regulate gene expression depending on electrode potential and lifestyle

Marinobacter spp. are opportunitrophs with a broad metabolic range including interactions with metals and electrodes. Marinobacter atlanticus strain CP1 was previously isolated from a cathode biofilm microbial community enriched from a sediment microbial fuel cell. Like other Marinobacter spp., M. atlanticus generates small amounts of electrical current when grown as a biofilm on an electrode, which is enhanced by the addition of redox mediators. However, the molecular mechanism resulting in extracellular electron transfer is unknown. Here, RNA-sequencing was used to determine changes in gene expression in electrode-attached and planktonic cells of M. atlanticus when grown at electrode potentials that enable current production (310 and 510 mV vs. SHE) compared to a potential that enables electron uptake (160 mV). Cells grown at current-producing potentials had increased expression of genes for molybdate transport, regardless of planktonic or attached lifestyle. Electrode-attached cells at current-producing potentials showed increased expression of the major export protein for the type VI secretion system. Growth at 160 mV resulted in an increase in expression of genes related to stress response and DNA repair including both RecBCD and the LexA/RecA regulatory network, as well as genes for copper homeostasis. Changes in expression of proteins with PEP C-terminal extracellular export motifs suggests that M. atlanticus is remodeling the biofilm matrix in response to electrode potential. These results improve our understanding of the physiological adaptations required for M. atlanticus growth on electrodes, and suggest a role for metal acquisition, either as a requirement for metal cofactors of redox proteins or as a possible electron shuttling mechanism.

Tonoplast-Localized OsMOT1;2 Participates in Interorgan Molybdate Distribution in Rice

Molybdenum (Mo) is an essential element for plant growth and is utilized by several key enzymes in biological redox processes. Rice assimilates molybdate ions via OsMOT1;1, a transporter with a high affinity for molybdate. However, other systems involved in the molecular transport of molybdate in rice remain unclear. Here, we characterized OsMOT1;2, which shares amino acid sequence similarity with AtMOT1;2 and functions in vacuolar molybdate export. We isolated a rice mutant harboring a complete deletion of OsMOT1;2. This mutant exhibited a significantly lower grain Mo concentration than the wild type (WT), but its growth was not inhibited. The Mo concentration in grains was restored by the introduction of WT OsMOT1;2. The OsMOT1;2-GFP protein was localized to the vacuolar membrane when transiently expressed in rice protoplasts. At the reproductive growth stage of the WT plant, OsMOT1;2 was highly expressed in the 2nd and lower leaf blades and nodes. The deletion of OsMOT1;2 impaired interorgan Mo allocation in aerial parts: relative to the WT, the mutant exhibited decreased Mo levels in the 1st and 2nd leaf blades and grains but increased Mo levels in the 2nd and lower leaf sheaths, nodes and internodes. When the seedlings were exposed to a solution with a high KNO3 concentration in the absence of Mo, the mutant exhibited significantly lower nitrate reductase activity in the shoots than the WT. Our results suggest that OsMOT1;2 plays an essential role in interorgan Mo distribution and molybdoenzyme activity in rice.

Pseudomonas aeruginosa T6SS-mediated molybdate transport contributes to bacterial competition during anaerobiosis.

Type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria and functions as a versatile protein export machinery that translocates effectors into eukaryotic or prokaryotic target cells. Growing evidence indicates that T6SS can deliver several effectors to promote bacterial survival in harmful environments through metal ion acquisition. Here, we report that the Pseudomonas aeruginosa H2-T6SS mediates molybdate (MoO42-) acquisition by secretion of a molybdate-binding protein, ModA. The expression of H2-T6SS genes is activated by the master regulator Anr and anaerobiosis. We also identified a ModA-binding protein, IcmP, an insulin-cleaving metalloproteinase outer membrane protein. The T6SS-ModA-IcmP system provides P.aeruginosa with a growth advantage in bacterial competition under anaerobic conditions and plays an important role in bacterial virulence. Overall, this study clarifies the role of T6SS in secretion of an anion-binding protein, emphasizing the fundamental importance of this bacterium using T6SS-mediated molybdate uptake to adapt to complex environmental conditions.

Revealing the salinity adaptation mechanism in halotolerant bacterium Egicoccus halophilus EGI 80432T by physiological analysis and comparative transcriptomics.

Egicoccus halophilus EGI 80432T, a halotolerant bacterium isolated from a saline-alkaline soil, belongs to a member of the class Nitriliruptoria, which exhibits high adaptability to salt environments. At present, the detailed knowledge of the salinity adaptation strategies of Nitriliruptoria was limited except for one research by using comparative genomics analysis. Here, we investigated the salinity adaptation mechanism of E. halophilus EGI 80432T by comparative physiological and transcriptomic analyses. The results of physiological analyses showed that trehalose and glutamate were accumulated by salt stress and showed the maximum at moderate salinity condition. Furthermore, the contents of histidine, threonine, proline, and ectoine were increased with increasing salt concentration. We found that both 0% and 9% NaCl conditions resulted in increased expressions of genes involved in carbohydrate and energy metabolisms, but negatively affected the Na+ efflux, iron, and molybdate transport. Moreover, the high salt condition led to enhancement of transcription of genes required for the synthesis of compatible solutes, e.g., glutamate, histidine, threonine, proline, and ectoine, which agree with the results of physiological analyses. The above results revealed that E. halophilus EGI 80432T increased inorganic ions uptake and accumulated trehalose and glutamate in response to moderate salinity condition, while the salinity adaptation strategy was changed from a "salt-in-cytoplasm" strategy to a "compatible solute" strategy under high salinity condition. The findings in this study would promote further studies in salt tolerance molecular mechanism of Nitriliruptoria and provide a theoretical support for E. halophilus EGI 80432T's application in ecological restoration.Key Points• Salt stress affected gene expressions responsible for carbohydrate and energy metabolisms of E. halophilus EGI 8042T.• E. halophilus EGI 80432T significantly accumulated compatible solutes under salt stress.• E. halophilus EGI 80432T adopted a "compatible solute" strategy to withstand high salt stress.

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

Selective Biosynthesis of Furoic Acid From Furfural by Pseudomonas Putida and Identification of Molybdate Transporter Involvement in Furfural Oxidation.

Upgrading of furanic aldehydes to their corresponding furancarboxylic acids has received considerable interest recently. Herein we reported selective oxidation of furfural (FAL) to furoic acid (FA) with quantitative yield using whole-cells of Pseudomonas putida KT2440. The biocatalytic capacity could be substantially promoted through adding 5-hydroxymethylfurfural into media at the middle exponential growth phase. The reaction pH and cell dosage had notable impacts on both FA titer and selectivity. Based on the validation of key factors for FAL conversion, the capacity of P. putida KT2440 to produce FAL was substantially improved. In batch bioconversion, 170 mM FA was produced with selectivity nearly 100% in 2 h, whereas 204 mM FA was produced with selectivity above 97% in 3 h in fed-batch bioconversion. Particularly, the role of molybdate transporter in oxidation of FAL and 5-hydroxymethylfurfural was demonstrated for the first time. The furancarboxylic acids synthesis was repressed markedly by destroying molybdate transporter, which implied Mo-dependent enzyme/molybdoenzyme played pivotal role in such oxidation reactions. This research further highlights the potential of P. putida KT2440 as next generation industrial workhorse and provides a novel understanding of molybdoenzyme in oxidation of furanic aldehydes.

Integrated Analysis of Molybdenum Nutrition and Nitrate Metabolism in Strawberry.

Molybdenum (Mo) is a component of the Mo cofactor (Moco) of nitrate reductase (NR) and is therefore essential for nitrate metabolism. However, little is known about Mo deficiency phenotypes or about how physiological and molecular mechanisms of Mo uptake and transport influence nitrate uptake and utilization in strawberry. Here, we used physiological and cytological techniques to identify Mo deficiency phenotypes in strawberry. Seedlings cultured with MoO4 2- grew well and exhibited normal microstructure and ultrastructure of leaves and roots. By contrast, seedlings cultivated under Mo-deficient conditions showed yellow leaf blades and ultrastructural changes such as irregular chloroplasts and unclear membrane structures that were similar to the symptoms of nitrogen deficiency. We cloned and analyzed a putative molybdate transporter, FaMOT1, which may encode a molybdate transporter involved in the uptake and translocation of molybdate. Interestingly, the addition of the molybdate analog tungstate led to lower tissue Mo concentrations, reduced the translocation of Mo from roots to shoots, and increased the plants’ sensitivity to Mo deficiency. Seedlings cultivated with MoO4 2- altered expression of genes in Moco biosynthesis. As expected, NR activity was higher under sufficient MoO4 2- levels. Furthermore, seedlings grown on Mo-deficient medium exhibited decreased 15NO3 - translocation and lower 15NO3 - use efficiency. These findings represent an important step towards understanding how molybdate transport, concentration, and deficiency symptoms influence nitrate uptake and utilization in strawberry.

Why does sulfate inhibit selenate reduction: Molybdenum deprivation from Mo-dependent selenate reductase

Selenium pollution has become an increasingly serious global concern. Methane-fed selenate reduction has proven to be of great interest for the bioremediation of selenate-contaminated waters even with the coexistence of nitrate and dissolved oxygen. However, it is unclear if the common concurrent sulfate anion affects selenate removal. To address this question, we first introduced selenate (SeO42-) as the sole influent electron acceptor in a CH4-fed membrane biofilm reactor (CH4-MBfR); then we added different concentrations of sulfate (SO42-). The initial selenate removal efficiency (∼90%) was decreased by 50% in the presence of 15.6 μM of sulfate and completely inhibited after loading with 171.9 μM of sulfate. 16S rRNA gene sequencing showed that the selenate-reducing bacteria decreased after the addition of sulfate. Metagenomic sequencing showed that the abundance of genes encoding molybdenum (Mo)-dependent selenate reductase reduced by >50% when exposed to high concentrations of sulfate. Furthermore, the decrease in the total genes encoding all Mo-oxidoreductases was much greater than that of the genes encoding molybdate transporters, suggesting that the inhibition of selenate reduction by sulfate was most likely via the direct competition with molybdate for the transport system, leading to a lack of available Mo for Mo-dependent selenate reductases and thus reducing their activities. This result was confirmed by a batch test wherein the supplementation of molybdate mitigated the sulfate effect. Overall, this study shed light on the underlying mechanism of sulfate inhibition on selenate reduction and laid the foundation for applying the technology to practical wastewaters.

Genetic mapping of ionomic quantitative trait loci in rice grain and straw reveals OsMOT1;1 as the putative causal gene for a molybdenum QTL qMo8.

Rice is a major dietary source of essential mineral nutrients and toxic elements (aka ionome) for humans. However, the genetic basis underlying the variation in ionome is still largely unknown. Here, we mapped 51 and 61 quantitative trait loci (QTLs) controlling the concentrations of 13 and 15 elements in rice (Oryza sativa L.) grain and straw, respectively, using a recombinant inbred lines (RILs) that were grown at three different field sites in 3years. Several QTLs were repeatedly detected in both grain and straw or in multiple years; the resulting 87 unique QTLs with 17 of them (20%) were co-localized with previously reported corresponding QTLs and 70 were novel ionomic QTLs. At least, 14 genomic clusters that controlled the concentrations of multiple elements were identified. Furthermore, we identified a molybdate transporter gene OsMOT1;1 as the putative causal gene for a QTL controlling molybdenum concentration in both straw and grain. QTL analyses based on the concentrations of multiple elements in both grain and straw of RIL population grown in three field sites in 3years allow us to identify tissue common QTLs and reproducible QTLs that were validated in multiple years. The identification of ionomic QTLs will be useful in revealing the molecular mechanisms underlying the accumulation of elements in rice and providing the opportunity to reduce the accumulation of toxic elements and enrich the accumulation of beneficial elements in rice grain.

Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.).

Plant–rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study’s main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover (Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation (EFD) and molybdate transporter 1 (MOT1). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.

Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame deletion of the modABC genes abolished the nicotine-degrading ability of strain J5, and complementation with modABC either from P. putida or Arthrobacter oxidans restored the degrading activity of the mutant to wild-type level. Nicotine degradation of J5 was inhibited markedly by addition of tungstate, a specific antagonist of molybdate. Molybdate at a non-physiologically high concentration (100 μM) fully restored nicotine-degrading activity and recovered growth of the modABC mutant in a nicotine minimal medium. Transcriptional analysis revealed that the expression of modABC was up-regulated at low molybdate concentrations and down-regulated at high moybdate concentrations, which indicated that at least one other system was able to transport molybdate, but with lower affinity. These results suggested that the molybdate transport system was essential to nicotine metabolism in P. putida J5.

Natural variation in a molybdate transporter controls grain molybdenum concentration in rice.

Molybdenum (Mo) is an essential micronutrient for most living organisms, including humans. Cereals such as rice (Oryza sativa) are the major dietary source of Mo. However, little is known about the genetic basis of the variation in Mo content in rice grain. We mapped a quantitative trait locus (QTL) qGMo8 that controls Mo accumulation in rice grain by using a recombinant inbred line population and a backcross introgression line population. We identified a molybdate transporter, OsMOT1;1, as the causal gene for this QTL. OsMOT1;1 exhibits transport activity for molybdate, but not sulfate, when heterogeneously expressed in yeast cells. OsMOT1;1 is mainly expressed in roots and is involved in the uptake and translocation of molybdate under molybdate-limited condition. Knockdown of OsMOT1;1 results in less Mo being translocated to shoots, lower Mo concentration in grains and higher sensitivity to Mo deficiency. We reveal that the natural variation of Mo concentration in rice grains is attributed to the variable expression of OsMOT1;1 due to sequence variation in its promoter. Identification of natural allelic variation in OsMOT1;1 may facilitate the development of rice varieties with Mo-enriched grain for dietary needs and improve Mo nutrition of rice on Mo-deficient soils.

Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum.

Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N2 reduction to NH3 is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron-molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo-free nitrogenases that are less efficient at reducing N2 than Mo-nitrogenase. To permit biogenesis of Mo-nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high-affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo-nitrogenase genes, while it represses production of Mo-free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo-mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo-responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.

The Molybdenum Storage Protein: A soluble ATP hydrolysis-dependent molybdate pump.

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 μmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied β-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules.

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth compared with wild-type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.

Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency.

Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotianatabacum (Tnt1) insertional mutant line were carried out in the one M.truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M.truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase.

LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild-type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp292 to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182nm. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons-removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L.japonicus for Mo uptake from the soil and growth, but is not for Mo delivery to the nodules.

Effect of exogenously applied molybdenum on its absorption and nitrate metabolism in strawberry seedlings

Molybdenum (Mo)—an essential element of plants—is involved in nitrogen (N) metabolism. Plants tend to accumulate more nitrate and show lower nitrogen use efficiency (NUE) under Mo-deficient conditions. Improving NUE in fruits reduces the negative effect of large applications of chemical fertilizer, but the mechanisms underlying how Mo enhances NUE remain unclear. We cultivated strawberry seedlings sprayed with 0, 67.5, 135, 168.75, or 202.5 g Mo·ha−1 in a non-soil culture system. The Mo concentration in every plant tissue analyzed increased gradually as Mo application level rose. Mo application affected iron, copper, and selenium adsorption in roots. Seedlings sprayed with 135 g Mo·ha−1 had a higher [15N] shoot:root (S:R) ratio, and 15NUE, and produced higher molybdate transporter type 1 (MOT1) expression levels in the roots and leaves. Seedlings sprayed with 135 g Mo·ha−1 also had relatively high nitrogen metabolic enzyme activities and up-regulated transcript levels of nitrate uptake genes (NRT1.1; NRT2.1) and nitrate-responsive genes. Furthermore, there was a significantly lower NO3− concentration in the leaves and roots, a higher NH4+ concentration in leaves, and a higher glutamine/glutamate (Gln/Glu) concentration at 135 g Mo·ha−1. Seedlings sprayed with 202.5 g Mo·ha−1 showed the opposite trend. Taken together, these results suggest that a 135 g Mo·ha−1 application was optimal because it enhanced NO3− transport from the roots to the shoots and increased NUE by mediating nitrogen metabolic enzyme activities, nitrate transport, and nitrate assimilation gene activities.

Non-Specific Root Transport of Nutrient Gives Access to an Early Nutritional Indicator: The Case of Sulfate and Molybdate

Under sulfur (S) deficiency, crosstalk between nutrients induced accumulation of other nutrients, particularly molybdenum (Mo). This disturbed balanced between S and Mo could provide a way to detect S deficiency and therefore avoid losses in yield and seed quality in cultivated species. Under hydroponic conditions, S deprivation was applied to Brassica napus to determine the precise kinetics of S and Mo uptake and whether sulfate transporters were involved in Mo uptake. Leaf contents of S and Mo were also quantified in a field-grown S deficient oilseed rape crop with different S and N fertilization applications to evaluate the [Mo]:[S] ratio, as an indicator of S nutrition. To test genericity of this indicator, the [Mo]:[S] ratio was also assessed with other cultivated species under different controlled conditions. During S deprivation, Mo uptake was strongly increased in B. napus. This accumulation was not a result of the induction of the molybdate transporters, Mot1 and Asy, but could be a direct consequence of Sultr1.1 and Sultr1.2 inductions. However, analysis of single mutants of these transporters in Arabidopsis thaliana suggested that other sulfate deficiency responsive transporters may be involved. Under field conditions, Mo content was also increased in leaves by a reduction in S fertilization. The [Mo]:[S] ratio significantly discriminated between the plots with different rates of S fertilization. Threshold values were estimated for the hierarchical clustering of commercial crops according to S status. The use of the [Mo]:[S] ratio was also reliable to detect S deficiency for other cultivated species under controlled conditions. The analysis of the leaf [Mo]:[S] ratio seems to be a practical indicator to detect early S deficiency under field conditions and thus improve S fertilization management.