OsNPF5.16, a nitrate transporter gene with natural variation, is essential for rice growth and yield

Rice has a large number of nitrate or peptide transporter family(NPF) genes, but the effects of most members on rice growth and development are unknown. We report that Os NPF5.16, a nitrate transporter gene with natural variation in its promoter sequence, is essential for rice growth and yield. The promoter sequence showed various differences between indica and japonica cultivars, and higher expression of Os NPF5.16 was found in indica cultivars with higher plant weight and more tillers than japonica cultivars.Os NPF5.16 was highly expressed in roots, tiller basal parts, and leaf sheaths, and its protein was localized on the plasma membrane. In c RNA-injected Xenopus laevis oocytes, Os NPF5.16 transport of nitrate at high nitrate concentration depended on p H. Overexpression of Os NPF5.16 increased nitrate content and total nitrogen content in leaf sheath as well as biomass and tiller bud length in rice. Elevated expression of Os NPF5.16 increased rice tiller number and grain yield by regulating cytokinin levels. Inhibition of Os NPF5.16 expression showed the opposite effects. Regulating Os NPF5.16 expression has potential for improving rice grain yield.

Influences of external nutrient conditions on the transcript levels of a nitrate transporter gene in Skeletonema costatum

To verify the feasibility of high-affinity nitrate transporter gene （Nrt2） as an indicator of nitrogen status, changes in the transcript levels of transcripts associated with phosphate starvation and different nitrate concentrations were studied using real-time quantitative reverse-transcription PCR （QRT-PCR） technology in batch cultures of Skeletonema costatum. The results show that compared with P-replete condition, P starvation could reduce the Nrt2 transcript levels apparently. Nrt2 transcript levels had a significant negative linear correlation with nitrate concentrations below 40 pmol/L. The results of 48 h short-term incubation experiment under different nitrate concentrations confirmed this correlation, and the following regression equation is built： y = -3.305x ＋ 98.95, R2 = 0.988, where x represents nitrate concentrations （〈40 btmol/L） and y represents the Nrt2 transcript levels.

Improving Nitrogen Use Efficiency in Rice through Enhancing Root Nitrate Uptake Mediated by a Nitrate Transporter, NRT1.1B

Nitrogen （N） is one of most important nutrients for crop production, which makes up 1%-5% of total plant dry matter （Marschner, 2012）. Due to the limited availability of N in soil, application of N fertilizers has been an important agronomic practice to increase crop yield. However, over-application of N fertilizers has caused pollution of N in soil, water and air. It was estimated that the nitrogen use efficiency （NUE, the total biomass or grain yield produced per unit of applied fertilizer N） in cereal crops is as low as 33% （Raun and Johnson, 1999）. Therefore, improving NUE together with reducing application of N fertilizers is an important issue for environment and sustainable production of crops. This is especially important for rice, which is a staple food for half population in the world.

Nitrate leaching of winter wheat grown in lysimeters as affected by fertilizers and irrigation on the North China Plain

Proper application of nitrogen（N） fertilizers and irrigation management are important production practices that can reduce nitrate leaching into groundwater and improve the N use efficiency（NUE）. A lysimeter/rain shelter facility was used to study effects of the rate of N fertilization, type of N fertilizer, and irrigation level on key aspects of winter wheat production over three growing seasons（response variables were nitrate transport, N leaching, and NUE）. Results indicated that nitrate concentration in the soil profile and N leaching increased with the rate of N fertilization. At the end of the third season, nitrate concentration in the top 0–75 cm layer of soil was higher with manure treatment while urea treatments resulted in higher concentrations in the 100–200 cm layer. With normal irrigation, 3.4 to 15.3% of N from applied fertilizer was leached from the soil, yet no leaching occurred under a stress irrigation treatment. The manure treatment experienced less N leaching than the urea treatment in all cases except for the 180 kg N ha^-1 rate in 2011–2012（season 3）. In terms of grain yield（GY）, dry matter（DM） or NUE parameters, values for the manure treatment were lower than for the urea treatment in 2009–2010（season 1）, yet were otherwise higher for urea treatment in season 3. GY and crop nitrogen uptake（NU） were elevated when the rate of N fertilizer increased, while the NUE decreased; GY, DM, and NU increased with the amount of irrigation. Data indicated that reduced rates of N fertilization combined with increased manure application and proper irrigation management can lower nitrate levels in the subsoil and reduce potential N leaching into groundwater.

Characterization and map-based cloning of miniature2-m1, a gene controlling kernel size in maize

Kernel development plays an important role in determining kernel size in maize.Here we present the cloning and characterization of a maize gene,nitrate transporter1.5(NRT1.5),which controls small kernel phenotype by playing an important role in kernel development.A novel recessive small kernel mutant miniature2-m1(mn2-m1)was isolated from self-pollinated progenies of breeding materials.The mutant spontaneously showed small kernel character arresting both embryo and endosperm development at an early stage after pollination.Utilizing 21 polymorphic SSR markers,the mn2-m1 locus was limited to a 209.9-kb interval using 9176 recessive individuals of a BC1 segregating population from mn2-m1/B73.Only one annotated gene was located in this 209.9 kb region,Zm00001 d019294,which was predicted to encode nitrate transporter1.5(NRT1.5).Allelism tests confirmed that mn2-m1 was allelic to miniature2-m2(mn2-m2)and miniature2-710 B(mn2-710 B).The mn2-m1 and mn2-m2 alleles both had nucleotide deletions in the coding region resulting in premature termination,and the mn2-710 B allele had some missence mutations.Subcellular localization showed that Miniature 2(MN2)is localized in the plasma membrane.Quantitative real-time PCR(qRT-PCR)analysis revealed that the expression of MN2 and some genes involved in the basal endosperm transfer layer(BETL)and embryo surrounding region(ESR)development were affected in mn2-m1 seeds.These results suggested that MN2 plays an important role in maize seed development.

Brassinosteroids modulate nitrogen physiological response and promote nitrogen uptake in maize(Zea mays L.)

Brassinosteroids(BRs)are steroid hormones that function in plant growth and development and response to environmental stresses and nutrient supplies.However,few studies have investigated the effect of BRs in modulating the physiological response to nitrogen(N)supply in maize.In the present study,BR signalingdeficient mutant zmbri1-RNAi lines and exogenous application of 2,4-epibrassinolide(e BL)were used to study the role of BRs in the regulation of physiological response in maize seedlings supplied with N.Exogenous application of e BL increased primary root length and plant biomass,but zmbri1 plants showed shorter primary roots and less plant biomass than wild-type plants under low N(LN)and normal N(NN)conditions.LN induced the expression of the BR signaling-associated genes Zm DWF4,Zm CPD,Zm DET2,and Zm BZR1 and the production of longer primary roots than NN.Knockdown of Zm BRI1 weakened the biological effects of LN-induced primary root elongation.e BL treatment increased N accumulation in shoots and roots of maize seedlings exposed to LN or NN treatment.Correspondingly,zmbri1 plants showed lower N accumulation in shoots and roots than wild-type plants.Along with reduced N accumulation,zmbri1 plants showed lower NO3-fluxes and^(15)NO_(3)^(-)uptake.The expression of nitrate transporter(NRT)genes(Zm NPF6.4,Zm NPF6.6,Zm NRT2.1,Zm NRT2.2)was lower in zmbri1 than in wild-type roots,but e BL treatments up-regulated the transcript expression of NRT genes.Thus,BRs modulated N physiological response and regulated the transcript expression of NRT genes to promote N uptake in maize.

Simulation of water and nitrogen dynamics as affected by drip fertigation strategies

The aim of drip fertigation is synchronising the application of water and nutrients with crop requirements, and maintaining the proper concentration and distribution of nutrient and water in the soil. The wetting patterns and nutrient distributions under drip fertigation have been proved to be closely related to the fertigation strategies. In order to find out the critical factors that affect the nutrient distribution under different drip fertigaiton strategies, a computer simulation model HYDRUS2D/3D was used to simulate the water and nitrate distribution for various fertigation strategies from a surface point source. Simulation results were compared with the observed ones from our previous studies. A 15° wedge-shaped plexiglass container was used in our experiment to represent one-twenty-fourth of the complete cylinder. The height of container is 40 cm, and the radius is 41 cm. The ammonium nitrate solution was added through a no. 7 needle connected to a Mariotte tube with a flexible hose. The soil water content, nitrate and ammonium concentrations were measured. The comparison of simulated and observed data demonstrated that the model performed reliably. The numerical analysis for various fertigation strategies from a surface point source showed that：（1） The total amount of irrigation water, the concentration of the fertilizer solution and the amount of pure water used to flush the pipeline after fertilizer solution application are the three critical factors influencing the distribution of water and fertilizer nitrogen in the soil.（2） The fresh water irrigation duration prior to fertigation has no obvious effect on nitrate distribution. The longer flushing time period after fertigation resulted in nitrate accumulation closer to the wetting front. From the point of avoiding the possibility of nitrate loss from the root zone, we recommended that the flushing time period should be as shorter as possible.（3） For a given amount of fertilizer, higher concentration of the fertilizer applied solution reduces the potential of nitrate leaching in drip irrigation system. While, lower concentration of the fertilizer solution resulted in an uniform distribution of nitrate band closer to the wetted front.

PuHox52 promotes coordinated uptake of nitrate,phosphate, and iron under nitrogen deficiency in Populus ussuriensis

It is of great importance to better understand how trees regulate nitrogen(N) uptake under N deficiency conditions which severely challenge afforestation practices, yet the underlying molecular mechanisms have not been well elucidated. Here,we functionally characterized PuHox52, a Populus ussuriensis HD-ZIP transcription factor, whose overexpression greatly enhanced nutrient uptake and plant growth under N deficiency. We first conducted an RNA sequencing experiment to obtain root transcriptome using PuHox52-overexpression lines of P. ussuriensis under low N treatment. We then performed multiple genetic and phenotypic analyses to identify key target genes of PuHox52 and validated how they acted against N deficiency under PuHox52 regulation.PuHox52 was specifically induced in roots by N deficiency, and overexpression of PuHox52promoted N uptake, plant growth, and root development. We demonstrated that several nitrate-responsive genes(PuNRT1.1, PuNRT2.4,PuCLC-b, PuNIA2, PuNIR1, and PuNLP1),phosphate-responsive genes(PuPHL1A and PuPHL1B), and an iron transporter gene(PuIRT1) were substantiated to be direct targets of PuHox52. Among them, PuNRT1.1, PuPHL1A/B, and PuIRT1 were upregulated to relatively higher levels during PuHox52-mediated responses against N deficiency in PuHox52-overexpression lines compared to WT. Our study revealed a novel regulatory mechanism underlying root adaption to N deficiency where PuHox52 modulated a coordinated uptake of nitrate, phosphate, and iron through 'PuHox52-PuNRT1.1', 'PuHox52-PuPHL1A/PuPHL1B', and'PuHox52-PuIRT1' regulatory relationships in poplar roots.

Nitrate Transport, Sensing, and Responses in Plants

Nitrogen （N） is an essential macronutrient that affects plant growth and development. N is an important component of chlorophyll, amino acids, nucleic acids, and secondary metabolites. Nitrate is one of the most abundant N sources in the soil. Because nitrate and other N nutrients are often limiting, plants have developed sophisticated mechanisms to ensure adequate supply of nutrients in a variable environment. Nitrate is absorbed in the root and mobilized to other organs by nitrate transporters. Nitrate sensing activates signaling pathways that impinge upon molecular, metabolic, physiological, and developmental responses locally and at the whole plant level. With the advent of genomics technologies and genetic tools, important advances in our understanding of nitrate and other N nutrient responses have been achieved in the past decade. Furthermore, techniques that take advantage of natural polymor- phisms present in divergent individuals from a single species have been essential in uncovering new components. However, there are still gaps in our understanding of how nitrate signaling affects biolog- ical processes in plants. Moreover, we still lack an integrated view of how all the regulatory factors iden- tified interact or crosstalk to orchestrate the myriad N responses plants typically exhibit. In this review, we provide an updated overview of mechanisms by which nitrate is sensed and transported throughout the plant. We discuss signaling components and how nitrate sensing crosstalks with hormonal pathways for developmental responses locally and globally in the plant. Understanding how nitrate impacts on plant metabolism, physiology, and growth and development in plants is key to improving crops for sustainable agriculture.

Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently, some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study Identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system （HATS） in the rice （Oryza sativa L. subsp, japonica cv. Nipponbare） genome. OsNRT2.1 and OsNRT2.2 share an Identical coding region sequence, and their deduced proteins are closely related to those from mono-cotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse trsnscription-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downrsgulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members. The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the Individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH （pH 8.0） inhibited nitrate influx, and acidic pH （pH 5.0） enhanced the nitrate influx in 1 h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

Nitrate is a major nitrogen （N） source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system （HATS） by exogenous abscisic acid （ABA） and glutamine （Gin） in wheat （Triticum aestivum L.） roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L ^15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.

Two NPF transporters mediate iron long-distance transport and homeostasis in Arabidopsis

Iron(Fe)transport and reallocation are essential to Fe homeostasis in plants,but it is unclear how Fe homeostasis is regulated,especially under stress.Here we report that NPF5.9 and its close homolog NPF5.8 redundantly regulate Fe transport and reallocation in Arabidopsis.NPF5.9 is highly upregulated in response to Fe deficiency.NPF5.9 expresses preferentially in vasculature tissues and localizes to the trans-Golgi network,and NPF5.8 showed a similar expression pattern.Long-distance Fe transport and allocation into aerial parts was significantly increased in NPF5.9-overexpressing lines.In the double mutant npf5.8 npf5.9,Fe loading in aerial parts and plant growth were decreased,which were partially rescued by Fe supplementation.Further analysis showed that expression of PYE,the negative regulator for Fe homeostasis,and its downstream target NAS4 were significantly altered in the double mutant.NPF5.9 and NPF5.8 were shown to also mediate nitrate uptake and transport,although nitrate and Fe application did not reciprocally affect each other.Our findings uncovered the novel function of NPF5.9 and NPF5.8 in long-distance Fe transport and homeostasis,and further indicated that they possibly mediate nitrate transport and Fe homeostasis independently in Arabidopsis.

An overview of emissions of SO2 and NOx and the long-range transport of oxidized sulfur and nitrogen pollutants in East Asia

The long-range transport of oxidized sulfur（sulfur dioxide（SO2） and sulfate） and oxidized nitrogen（nitrogen oxides（NOx ） and nitrate） in East Asia is an area of increasing scientific interest and political concern. This paper reviews various published papers, including ground- and satellite-based observations and numerical simulations. The aim is to assess the status of the anthropogenic emissions of SO2 and NOx and the long-range transport of oxidized S and N pollutants over source and downwind region. China has dominated the emissions of SO2 and NOx in East Asia and urgently needs to strengthen the control of their emissions, especially NOx emissions. Oxidized S and N pollutants emitted from China are transported to Korea and Japan, due to persistent westerly winds, in winter and spring.However, the total contributions of China to S and N pollutants across Korea and Japan were not found to be dominant over longer time scales（e.g., a year）. The source–receptor relationships for oxidized S and N pollutants in East Asia varied widely among the different studies. This is because：（1） the nonlinear effects of atmospheric chemistry and deposition processes were not well considered, when calculating the source–receptor relationships;（2） different meteorological and emission data inputs and solution schemes for key physical and chemical processes were used; and（3） different temporal and spatial scales were employed. Therefore, simulations using the same input fields and similar model configurations would be of benefit, to further evaluate the source–receptor relationships of the oxidized S and N pollutants.

Determination of nitrogen reduction levels necessary to reach groundwater quality targets in Slovenia

Within a collaborative project between Slovenian Environment Agency （ARSO） and Research Center Jfilich （FZJ）, nitrogen reduction levels necessary to reach groundwater quality targets in Slovenia were assessed. For this purpose the hydrological model GROWA- DENUZ was coupled with agricultural N balances and applied consistently to the whole territory of Slovenia in a spatial resolution of 100 x 100 m. GROWA was used to determine the water balance in Slovenia for the hydrologic period 1971-2000. Simultaneously, the displaceable N load in soft was assessed from agricultural Slovenian N surpluses for 2011 and the atmospheric N deposition. Subsequently, the DENUZ model was used to assess the nitrate degradation in soil and, in combination with the percolation water rates from the GROWA model, to determine nitrate concentration in the leachate. The areas showing predicted nitrate concentrations in the leachate above the EU groundwater quality standard of 50 mg NO3/L have been identified as priority areas for implementing nitrogen reduction measures. For these ＂hot spot＂ areas DENUZ was used in a backward mode to quantify the maximal permissible nitrogen surplus levels in agriculture to guarantee a nitrate concentration in percolation water below 50 mg NO3/L. Model results indicate that additional N reduction measures should be implemented in priority areas rather than area-covering. Research work will directly support the implementation of the European Union Water Framework Directive in Slovenia, e.g., by using the maximal permissible nitrogen surplus levels as a framework for the derivation of regionally adapted and hence effective nitrogen reduction measures.