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AKT2 potassium (K+) channels are members of the plant Shaker family which mediate dual-directional K+ transport with weak voltage-dependency. Here we show that OsAKT2 of rice (Oryza sativa) functions mainly as an inward rectifier with strong voltage-dependency and acutely suppressed outward activity. This is attributed to the presence of a unique K191 residue in the S4 domain. The typical bi-directional leak-like property was restored by a single K191R mutation, indicating that this functional distinction is an intrinsic characteristic of OsAKT2. Furthermore, the opposite R195K mutation of AtAKT2 changed the channel to an inward-rectifier similar to OsAKT2. OsAKT2 was modulated by OsCBL1/OsCIPK23, evoking the outward activity and diminishing the inward current. The physiological relevance in relation to the rectification diversity of OsAKT2 was addressed by functional assembly in the Arabidopsis (Arabidopsis thaliana) akt2 mutant. Overexpression (OE) of OsAKT2 complemented the K+ deficiency in the phloem sap and leaves of the mutant plants but did not significantly contribute to the transport of sugars. However, the expression of OsAKT2-K191R overcame both the shortage of phloem K+ and sucrose of the akt2 mutant, which was comparable to the effects of the OE of AtAKT2, while the expression of the inward mutation AtAKT2-R195K resembled the effects of OsAKT2. Additionally, OE of OsAKT2 ameliorated the salt tolerance of Arabidopsis., The presence of a unique K191 residue retains the activity of rice potassium channel OsAKT2 mainly as an inward rectifier (Mode I) that emphasizes its in planta role of phloem K+ translocation.

K+ uptake to root conducting cells is a prerequisite for its release to the xylem vessels and long-distance delivery to the aerial parts, though the molecular mechanism underpinning such process remains insufficiently understood. Here we report the discovery in rice root of an essential component involved in loading of K+ to the stelar tissues encoded by an unusual short Shaker K+ channel OsK2.2. OsK2.2 represents a novel type of short Shaker channels lacking the sequences immediately downstream of the putative cyclic nucleotide binding domain that present to the C-termini of all plant Shaker channels reported to date. In silico analysis suggests that such short Shaker channels are monocot-specific and especially found in the Poaceae species. Functional assessment in Xenopus oocytes reveals that OsK2.2 uses such innate C-terminal shortness as an intrinsic strategy for maintaining large capacity of K+ uptake to the cells. OsK2.2 predominantly localizes to the phloem and xylem peripheral cells of rice root vasculature. Disruption of OsK2.2 function causes approximately 30% lower K+ in both phloem and xylem sap and reduces K+ accumulation to the shoots. OsK2.2 strongly contributes to the grain yield of rice under field conditions. The essential contribution of such particular type of inward Shaker channel, operating in stelar tissues, to K+ transport between roots and shoots and to grain yield, here evidenced in rice, may be a common trait amongst Poaceae species.

Because of highly intensive farming practices, crop plants could suffer relatively long terms of ammonium (NH+4) excess stress introduced by overdose application of nitrogen fertilizers. However, the lack of sufficient understanding of plant responses to NH+4 excess stress impairs the detection of effective solutions to this problem. The present work examined the biological influences of over-supplied NH+4 in Arabidopsis thaliana using two mutant lines each with an ammonium transporter (AMT) gene (AtAMT1;1 or AtAMT1;3) knocked out. Our results indicated that lacking one of the major components of root NH+4 -absorbing systems significantly alleviated the toxicity effects on Arabidopsis plants by reducing the accumulation of free NH+4, suggesting that persistent absorption of NH+4 through AMT was the main cause of excessive accumulation of free NH+4 in the plants. Shading treatment led to a reduced transpirational driving force and thereby constrained the accumulation of toxic NH+4 in the plants, finally resulting in higher NH+4 -promoted growth in the wild type (WT). Under the shading treatment, the amt1;1 and amt1;3 mutant plants acquired insufficient NH+4 and showed reduced growth when compared with the WT. Furthermore, the foliar application of sucrose notably alleviated the inhibitory effects on plant growth in the WT but had no effect on either the amt1;1 or amt1;3 mutant plants, indicating that carbon scarcity associated with NH+4 excess is probably a major cause of NH+4 toxicity in plants. Accordingly, increasing carbon source could be a potentially effective approach that alleviates the inhibition caused by NH+4 excess and increases nitrogen use efficiency under NH+4 over-supply.

In aquatic fields, ammonium (NH4+) is the most preferred nitrogen (N) source used by plants. The uptake of NH4+ is facilitated by the family of ammonium transporters (AMTs). However, the molecular functions of AMTs in aquatic plants are largely unknown. In this work, a new NH4+ transporter encoding gene, ApAMT1;1, was isolated from the typical aquatic plant alligatorweed, using degenerated primers and rapid amplification of cDNA end (RACE) techniques. Quantitative real time PCR showed that ApAMT1;1 was predominantly expressed in roots, and significantly induced by NH4+ starvation in all tested tissues, including leaves, stems and roots. Functional determination and 15N-labeled ammonium uptake assays in yeast cells indicated that ApAMT1;1 was a typical high-affinity transporter, with a 38.6 μM Km value, and the phosphorylation site T469 was required to retain its NH4+ uptake capacity. Further analyses with Met sulfoximine (MSX), a NH4+ assimilation inhibitor, demonstrated that ApAMT1;1-mediated NH4+ uptake might be feedback regulated by the internal NH4+ accumulation. Our results reveal a functional role of ApAMT1;1 in the uptake and transport of NH4+ in aquatic plants.

Bipolar disorder (BD) is a severe psychiatric illness attributable to multifactorial risk components (e.g. environmental stimuli, neuroinflammation, etc.), and genetic variations affecting these risk components are considered pivotal predisposing factors. The interleukin-1β (IL-1β) gene and its protein product have been repeatedly highlighted in the pathogenesis of BD. As functional polymorphisms and haplotypes linked with IL-1β mRNA expression have been reported, whether they are correlated with the risk of developing BD remains to be tested.To examine whether variations in the IL-1β gene locus confer genetic risk of BD, we recruited 930 BD patients and 912 healthy controls for the current study. All subjects were Han Chinese, and were age- and gender-matched. We tested seven functional single nucleotide polymorphisms (SNPs) spanning the IL-1β gene and one haplotype composed of three SNPs for their associations with risk of BD.We found that the functional SNPs in the promoter region of IL-1β gene were significantly associated with risk of BD. The haplotype analyses further supported the involvement of IL-1β promoter SNPs in BD. The BD risk SNPs in our study have been previously reported to predict higher IL-1β levels in the brain and peripheral blood, which is consistent with the clinical observation of elevated IL-1β levels in the lymphocytes or peripheral blood of patients with BD compared with healthy subjects.Our results support the contention that IL-1β is likely a risk gene for BD, and further investigations on this gene may promote our understanding and clinical management of this illness.

Maize grows in nitrate-dominated dryland soils, but shortly upon localized dressing of nitrogen fertilizers, ammonium is retained as a noticeable form of nitrogen source available to roots. Thus in addition to nitrate, the absorption of ammonium can be an important strategy that promotes rapid plant growth at strong nitrogen demanding stages. The present study reports the functional characterization of three root-expressed ammonium transporters (AMTs), aiming at finding out functional and regulatory properties that correlate with efficient nitrogen acquisition of maize. Using a stable electrophysiological recording method we previously established in Xenopus laevis oocytes that integrates the capture of currents in response to voltage ramps with onsite stability controls, we demonstrate that all three ZmAMT1s engage NH4+ uniporting as ammonium uptake mechanisms. The Km value for ZmAMT1.1a, 1.1b, or ZmAMT1.3 is, respectively, 9.9, 15.6, or 18.6 μM, indicating a typical high-affinity transport of NH4+ ions. Importantly, the uptake currents of these ZmAMT1s are markedly amplified upon extracellular acidification. A pH drop from 7.4 to 5.4 results in a 140.8%, 64.1% or a 120.7% increase of ammonium uptake activity through ZmAMT1.1a, 1.1b, or ZmAMT1.3. Since ammonium uptake by plant roots accompanies a spontaneous acidification to the root medium, the functional promotion of ZmAMT1.1a, 1.1b, and ZmAMT1.3 by low pH is in line with the facilitated ammonium uptake activity in maize roots. Furthermore, the expression of the three ZmAMT1 genes is induced under ammonium-dominated conditions. Thus we describe a facilitated ammonium uptake strategy in maize roots by functional and expression regulations of ZmAMT1 transporters that may coordinate with efficient acquisition of this form of nitrogen source when available.

International audience; AtAMT1;3 is a major contributor to high-affinity ammonium uptake in Arabidopsis roots. Using a stable electrophysiological recording strategy, we demonstrate in Xenopus laevis oocytes that AtAMT1;3 functions as a typical high-affinity NH4 + uniporter independent of protons and Ca2+. The findings that AtAMT1;3 transports methylammonium (MeA+, a chemical analog of NH4 +) with extremely low affinity (K m in the range of 2.9-6.1 mM) led to investigate the mechanisms underlying substrate binding. Homologous modeling and substrate docking analyses predicted that the deduced substrate binding motif of AtAMT1;3 facilitates the binding of NH4 + ions but loosely accommodates the binding of MeA+ to a more superficial location of the permeation pathway. Amongst point mutations tested based on this analysis, P181A resulted in both significantly increased current amplitudes and substrate binding affinity, whereas F178I led to opposite effects. Thus these 2 residues, which flank W179, a major structural component of the binding site, are also important determinants of AtAMT1;3 transport capacity by being involved in substrate binding. The Q365K mutation neighboring the histidine residue H378, which confines the substrate permeation tunnel, affected only the current amplitudes but not the binding affinities, providing evidence that Q365 mainly controls the substrate diffusion rate within the permeation pathway.

Additional file 2: Figure S2. O2− localization in situ.

Additional file 1: Figure S1. H2O2 localization in situ.

Additional file 3: Figure S3. Free amino acid contents assays.

Background: Overuse of nitrogen fertilizers is often applied to satisfy strong nitrogen demand of high–yielding rice, leading to persistent NH4+ excess in the plant. However, the mechanisms constraining the effectiveness of elevated plant NH4+ in plant growth and grain yield of rice are not sufficiently addressed. The current study analyses the early performance of such internal NH4+ excess in rice, aiming at finding out constraints against compromised nitrogen use efficiency Results: By mimicking a rapid accumulation of plant NH4+ and an RNA-Seq analysis, the present work reveals that internal NH4+ excess in rice plant initiates a burst of reactive oxygen species (ROS) and triggers probably specifically the activation of glutathione transferase (GST)–mediated glutathione cycling for ROS cleavage. Meanwhile, the suppression of the expression of genes involved in photon caption and the activity of primary CO2 fixation enzymes (Rubisco), provides implications of a reduction in photosynthetic carbon income.Along the progress of NH4+/ROS stresses, enhanced energy–producing processes (carbon breakdown) take place as indicated by strong induction of glycolysis related genes to meet the demand of energy consuming activation of ROS–cleavaging systems. The development of these defensive reactions causes a sugar scarcity in the plant that accumulatively leads to growth inhibition. To the opposite direction, a sucrose feeding treatment to the roots renders the accumulation of free NH4+ and ROS, partly restores the activities of photosynthetic CO2 fixation and thus alleviates the scarcity in active carbon source. Conclusion: Our results indicate that carbon scarcity is probably a major constraint in rice plant that limits the performance of nitrogen under overuse of N fertilizers. Keywords: rice, NH4+ excess, ROS, GSH cycle, carbon scarcity, sucrose feeding.

Background Overuse of nitrogen fertilizers is often a major practice to ensure sufficient nitrogen demand of high–yielding rice, leading to persistent NH4+ excess in the plant. However, this excessive portion of nitrogen nutrient does not correspond to further increase in grain yields. For finding out the main constraints related to this phenomenon, the performance of NH4+ excess in rice plant needs to be clearly addressed beyond the well-defined root growth adjustment. The present work isolates an acute NH4+ excess condition in rice plant from causing any measurable growth change and analyses the initial performance of such internal NH4+ excess. Results We demonstrate that the acute internal NH4+ excess in rice plant accompanies readily with a burst of reactive oxygen species (ROS) and initiates the downstream reactions. At the headstream of carbon production, photon caption genes and the activity of primary CO2 fixation enzymes (Rubisco) are evidently suppressed, indicating a reduction in photosynthetic carbon income. Next, the vigorous induction of glutathione transferase (GST) genes and enzyme activities along with the rise of glutathione (GSH) production suggest the activation of GSH cycling for ROS cleavage. Third, as indicated by strong induction of glycolysis / glycogen breakdown related genes in shoots, carbohydrate metabolisms are redirected to enhance the production of energy and carbon skeletons for the cost of ROS scavenging. As the result of the development of these defensive reactions, a carbon scarcity would accumulatively occur and lead to a growth inhibition. Finally, a sucrose feeding cancels the ROS burst, restores the activity of Rubisco and alleviates the demand for the activation of GSH cycling. Conclusion Our results demonstrate that acute NH4+ excess accompanies with a spontaneous ROS burst and causes carbon scarcity in rice plant. Therefore, under overuse of N fertilizers carbon scarcity is probably a major constraint in rice plant that limits the performance of nitrogen.

International audience; Water deficit is one of the main environmental constraints that limit plant growth. Accordingly, plants evoke rather complex strategies to respond and/or acclimate to such frustrating circumstances. Due to insufficient understandings of acclimatory mechanisms of plants' tolerance to persistent water deficit, a desert shrub of an ancient origin, Ammopiptanthus mongolicus, has recently attracted growing attentions. Differed from Arabidopsis, the opening of stomata of A. mongolicus is constrained by low external K+ concentration of the guard cells. Although as a general consequence, a raised level of ABA is also induced in A. mongolicus following water deficit, this does not accordingly result in efficient stomatal closure. In consistent with this phenomenon, the expression of genes coding for the negative regulators of the ABA signaling cascade-the type 2C protein phosphatases (PP2Cs) are notably induced, whereas the transcription of the downstream SnRK2 protein kinase genes or the destination ion fluxing channel genes remain almost unaffected under water deficit treatments. Therefore, in term of stomatal control in response to water deficit, A. mongolicus seemingly employs an unusual strategy: a constrained stomatal opening controlled by extracellular K+ concentrations rather than a prompt stomatal closure triggered by ABA-induced signaling pathway. Additionally, an acute accumulation of proline is induced by water deficit which may partly compromise the activation of antioxidant enzymes in A. mongolicus. Such strategy of stomatal control found in A. mongolicus may in certain extents, reflect the acclimatory divergence for plants' coping with persistent stress of water deficit.

Ammonium transporter (AMT)-mediated acquisition of ammonium nitrogen from soils is essential for the nitrogen demand of plants, especially for those plants growing in flooded or acidic soils where ammonium is dominant. Recent advances show that AMTs additionally participate in many other physiological processes such as transporting ammonium from symbiotic fungi to plants, transporting ammonium from roots to shoots, transferring ammonium in leaves and reproductive organs, or facilitating resistance to plant diseases via ammonium transport. Besides being a transporter, several AMTs are required for the root development upon ammonium exposure. To avoid the adverse effects of inadequate or excessive intake of ammonium nitrogen on plant growth and development, activities of AMTs are fine-tuned not only at the transcriptional level by the participation of at least four transcription factors, but also at protein level by phosphorylation, pH, endocytosis, and heterotrimerization. Despite these progresses, it is worth noting that stronger growth inhibition, not facilitation, unfortunately occurs when AMT overexpression lines are exposed to optimal or slightly excessive ammonium. This implies that a long road remains towards overcoming potential limiting factors and achieving AMT-facilitated yield increase to accomplish the goal of persistent yield increase under the present high nitrogen input mode in agriculture.

Ammonium is the main inorganic nitrogen source in paddy soil. Rice (Oryza sativa), an ammonium-preferring and -tolerant grain crop, is a valuable resource for researching ammonium-uptake mechanism and understanding the molecular networks that the plant copes with ammonium variation. To generate a broad survey of early responses affected by varied ammonium supplies in rice, RNA samples were prepared from the roots and shoots of rice plants subjected to nitrogen-free (0mM ammonium), 1mM ammonium and high ammonium (10mM ammonium) for a short period of 4h (1mM ammonium treatment as the control), respectively, and the transcripts were sequenced using the Illumina/HiSeq™ 2000 RNA sequencing (RNA-Seq) platform. By comparative analysis, 394 differentially expressed genes (DEGs) were identified in roots, among which, 143 and 251 DEGs were up- and down-regulated under nitrogen-free condition, respectively. In shoots, 468 (119 up-regulated/349 down-regulated) DEGs were found under such condition. However, with high ammonium treatment, only 63 genes (6 up-regulated/57 down-regulated) in roots and 115 genes in shoots (93 up-regulated/22 down-regulated) were differentially expressed. According to KEGG analysis, when exposed to nitrogen-free condition, DEGs participating in the carbohydrate and amino acid metabolisms were down-regulated (with 1 exception) in roots as well as in shoots, implying reduced carbohydrate and nitrogen metabolisms. Under high ammonium supply, all DEGs associated with carbohydrate and amino acid metabolisms were down-regulated in roots and to the contrary, up-regulated in shoots. Aldehyde dehydrogenase (ALDH, NAD(+)) [EC: 1.2.1.3] seemed to have played an important role in rice shoots under high ammonium condition, analysis results implicated a coordinative regulation of carbohydrate with amino acid metabolisms under nitrogen deficiency as well as the high ammonium conditions during a short period of several hours in rice. Moreover, transcripts with abundance variation might be precious gene resources in responding to different ammonium supplies in rice.

ApKUPs are typical high-affinity potassium (K+) transporters of Alternanthera philoxeroides which are involved in its response to K+ starvation and abiotic stresses. In this study, the overexpression of ApKUP3 gene in rice resulted in enhanced K+ nutrition and drought tolerance of transgenic plants. Compared with wild-type (WT) plants, the transgenic plants showed a better growth performance and a strengthened K+ accumulation under different K+ supplies. The ApKUP3 overexpression in the rice plants also enhanced tolerance to a drought stress, as evidenced by a reduced leaf water loss and an increased total leaf chlorophyll content, stomatal conductance, net photosynthetic rate, and activities of superoxide dismutase, peroxidase, and ascorbate peroxidase (APX). Moreover, the transcription of genes involved in the antioxidation defense system were higher in the transgenic plants than in the WT plants upon the drought stress.

Ammopiptanthus mongolicus, an evergreen broadleaf legume shrub, can survive under conditions of high and low temperature, extreme salinity, and drought. This attribute makes it an ideal model for studying mechanisms of stress tolerance in plants. However, simple sequence repeat (SSR) resources for this species are insufficient in public databases. In this study, a total of 44,959 unigenes identified from the A. mongolicus transcriptome were used for SSR analysis by MIcroSAtellite (MISA). A total of 13,859 SSRs were found to be distributed within 10,409 unigenes, with an average length of 15 bp and an average density of one SSR per 4.4 kb. There were 222 different motif types in the A. mongolicus transcriptome, and mononucleotide repeats represented the main type, accounting for 44.2% of all SSRs. The (A/T)n repeat was the most frequent motif, accounting for 42.37% of all SSRs. We also performed Gene Ontology functional analysis, Kyoto Encyclopedia of Genes and Genomes database pathway analysis, and eggNOG analysis, and identified 6157, 2301, and 9845 unigenes containing SSRs in these three databases, respectively. The functional categorization of A. mongolicus unigenes containing SSRs revealed that these unigenes represent many transcribed genes with different functions. These data provide sequence information that may be used to improve molecular-assisted markers for the study A. mongolicus genetic diversity.

Ammonium transporters (AMTs) are major routes for plant uptake of the NH4+-form nitrogen. Plant AMTs mediate predominantly the uptake of NH4+ and to a lesser extent, its organic analog methylammonium (MeA+). Mutagenesis studies on potential phosphorylation residues have achieved solid recognition that alteration of the phosphorylation status can result in allosteric regulation and impair the functionality of plant AMTs. However, molecular insights to the fine-tuning of a functional ammonium transporter remain less clear. In this report, we demonstrate that the rice root expressed OsAMT1;3 (Oryza sativa ammonium transporter 1;3) functions as a typical high-affinity NH4+ transporter and is weakly permeable to MeA+ using growth assays in NH4+ uptake defective yeast cells and electrophysiological measurements in Xenopus oocytes. Upon screening of six point mutations generated with the transporter, we identified two amino acid residues involved in the functional modulation of OsAMT1;3. The H199E mutation caused loss of transport activity whereas other five mutations retained the functionality of OsAMT1;3. Furthermore, the L56F mutation enabled respectively 5- and 3.5 -fold increased capability for NH4+ and MeA+ uptake with several-fold decreased affinity (Km) and accelerated Vmax values. Surprisingly, yeast cells expressing the L56F mutation shown increased Na+ toxicity leading to a speculation that enhanced Na+ permeation occurred with this mutation. The phenomenon was further supported by the observation of significant Na+ uptake current in oocytes. Our results seemingly support a speculation that the L56F mutation of OsAMT1;3 widens the substrate passage tunnel and allows enhanced permeability to NH4+ and larger ions MeA+ and Na+.

Functional identification of a relatively lower affinity ammonium transporter, OsAMT1;1, which is a proton-independent feedback regulated ammonium transporter in rice. Rice genome contains at least 12 ammonium transporters, though their functionality has not been clearly resolved. Here, we demonstrate the functional properties of OsAMT1;1 applying functional complementation and 15NH4 + uptake determination in yeast cells in combination with electrophysiological measurements in Xenopus oocytes. Our results show that OsAMT1;1 is a NH4 + transporter with relatively lower affinity to NH4 + (110–129 μM in oocytes and yeast cells, respectively). Under our experimental conditions, OsAMT1;1-mediated NH4 + uptake or current is not significantly modulated by extra- or intracellular pH gradient, suggesting that this transporter probably functions as a NH4 + uniporter. Inhibition of yeast growth or currents elicited from oocytes by ammonium assimilation inhibitor l-methionine sulfoximine indicates that NH4 + transport by OsAMT1;1 is likely feedback regulated by accumulation of the substrate. In addition, effects of phosphorylation inhibitors imply that NH4 + uptake by OsAMT1;1 is also modulated by tyrosine-specific protein kinase or calcium-regulated serine/threonine-specific protein phosphatase involved phosphorylation processes.

UNLABELLED • PREMISE OF THE STUDY The potassium cation (K(+)), one of the most abundant cations in cells, improves plant tolerance to various abiotic stresses. Alligatorweed (Alternanthera philoxeroides) is well known for its strong capacity to accumulate K(+) The distinctive K(+) accumulation capability of alligatorweed is linked to a high-affinity K(+) transport facilitated by K(+)-uptake transporters (ApKUPs).• METHODS A putative K(+) transporter gene, ApKUP4, was isolated from alligatorweed using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Gene expression profiles were performed by quantitative real time PCR and northern blot analysis. Moreover, we introduced ApKUP4 into Arabidopsis to determine its function in improving crop nutrition and NaCl stress tolerance.• KEY RESULTS ApKUP4 was localized throughout the entire alligatorweed plant, and its expression was stimulated in the stems and roots under K(+) deficiency, osmotic stress, and salinity stress. Northern blot analysis revealed that ApKUP4 was present in all tested organs of transgenic Arabidopsis plants. Compared with the wild type, Arabidopsis plants overexpressing ApKUP4 showed improved growth and K(+) homeostasis. Moreover, ApKUP4 overexpression in Arabidopsis plants enhanced plant tolerance to salinity stress, as evidenced by reduced water loss and ROS generation, associated with enhanced photosynthesis, nutritional status, and enzymatic antioxidants.• CONCLUSIONS The present study provides direct evidence that the alligatorweed K(+) transporter gene, ApKUP4, contributes to salinity tolerance in transgenic Arabidopsis seedlings, demonstrating the essentiality of potassium homeostasis for plant salinity tolerance.