The rectification control and physiological relevance of potassium channel OsAKT2.

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 functional polymorphisms linked with interleukin-1ß gene expression are associated with bipolar disorder.

BACKGROUND: 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-1ß (IL-1ß) gene and its protein product have been repeatedly highlighted in the pathogenesis of BD. As functional polymorphisms and haplotypes linked with IL-1ß mRNA expression have been reported, whether they are correlated with the risk of developing BD remains to be tested. METHODS: To examine whether variations in the IL-1ß 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-1ß gene and one haplotype composed of three SNPs for their associations with risk of BD. RESULTS: We found that the functional SNPs in the promoter region of IL-1ß gene were significantly associated with risk of BD. The haplotype analyses further supported the involvement of IL-1ß promoter SNPs in BD. The BD risk SNPs in our study have been previously reported to predict higher IL-1ß levels in the brain and peripheral blood, which is consistent with the clinical observation of elevated IL-1ß levels in the lymphocytes or peripheral blood of patients with BD compared with healthy subjects. CONCLUSION: Our results support the contention that IL-1ß is likely a risk gene for BD, and further investigations on this gene may promote our understanding and clinical management of this illness.

Functional and Regulatory Characterization of Three AMTs in Maize Roots.

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 K m 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.

Functional Characterization of the Arabidopsis Ammonium Transporter AtAMT1;3 With the Emphasis on Structural Determinants of Substrate Binding and Permeation Properties.

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.

Function and Regulation of Ammonium Transporters in Plants.

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.

Internal ammonium excess induces ROS-mediated reactions and causes carbon scarcity in rice.

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.

An unusual strategy of stomatal control in the desert shrub Ammopiptanthus mongolicus.

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.

Identification of structural elements involved in fine-tuning of the transport activity of the rice ammonium transporter OsAMT1;3.

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+.

The S1-S2 linker determines the distinct pH sensitivity between ZmK2.1 and KAT1.

Efficient stomatal opening requires activation of KAT-type K(+) channels, which mediate K(+) influx into guard cells. Most KAT-type channels are functionally facilitated by extracellular acidification. However, despite sequence and structural homologies, the maize counterpart of Arabidopsis KAT1 (ZmK2.1) is resistant to pH activation. To understand the structural determinant that results in the differential pH activation of these counterparts, we analysed chimeric channels and channels with point mutations for ZmK2.1 and its closest Arabidopsis homologue KAT1. Exchange of the S1-S2 linkers altered the pH sensitivity between the two channels, suggesting that the S1-S2 linker is essentially involved in the pH sensitivity. The effects of D92 mutation within the linker motif together with substitution of the first half of the linker largely resemble the effects of substitution of the complete linker. Topological modelling predicts that one of the two cysteines located on the outer face section of the S5 domain may serve as a potential titratable group that interacts with the S1-S2 linker. The difference between ZmK2.1 and KAT1 is predicted to be the result of the distance of the stabilized linkers from the titratable group. In KAT1, residue K85 within the linker forms a hydrogen bond with C211 that enables the pH activation; conversely, the linker of ZmK2.1 is distantly located and thus does not interact with the equivalent titration group (C208). Thus, in addition to the known structural contributors to the proton activation of KAT channels, we have uncovered a previously unidentified component that is strongly involved in this complex proton activation network.

The rice OsAMT1;1 is a proton-independent feedback regulated ammonium transporter.

KEY MESSAGE: 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 (15)NH4 (+) 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.

RNA-Seq analysis of differentially expressed genes in rice under varied nitrogen supplies.

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.

Heterologous expression of an alligatorweed high-affinity potassium transporter gene enhances salinity tolerance in Arabidopsis thaliana.

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.

Analysis of common genetic variants identifies RELN as a risk gene for schizophrenia in Chinese population.

UNLABELLED: Abstract Objectives. Several lines of evidence have shown that both RELN mRNA and protein are possibly down-regulated in the brain of schizophrenia patients. Recent association studies in European populations suggested RELN as a risk gene for schizophrenia. In this study, we test if RELN contributes to the risk of schizophrenia in Chinese population. Methods. We conducted case-control association analysis of 19 representative single nucleotide polymorphisms (SNPs) spanning the entire region of RELN in two independent Han Chinese samples from southwestern China (the Kunming sample and the Yuxi sample). Results. We identified six SNPs significantly associated with schizophrenia in the Kunming sample and four of them remained significant in the combined samples (the P values range from 0.006 to 4.0 × 10(-5)). Haplotype analysis also suggested significant associations for the haplotypes incorporating the six significant SNPs (global P < 1.0 × 10(-5)). Additionally, we also observed several other haplotypes (defined by a different set of SNPs) significantly associated with schizophrenia in the Kunming sample. However, the reported association of rs7341475 in Ashkenazi Jews was not significant in Han Chinese. CONCLUSIONS: Our findings demonstrate that RELN is a susceptibility gene for schizophrenia in Chinese population, and it is likely a common risk gene for schizophrenia in major populations worldwide.

[Association of RELN SNP rs7341475 with schizophrenia in the Chinese population].

Schizophrenia is a common and complex psychiatric disorder. Significant evidence has suggested that genetic factors play pivotal roles in the etiology of schizophrenia. More than 100 schizophrenia candidate genes have been reported; however, many of them do not have satisfactory replications among different populations. Among these genes, RELN is thought to be associated with schizophrenia in many populations, suggesting it is a real risk gene for this disorder. Identified in the GWAS study, single nucleotide polymorphism (SNP) rs7341475, located in intron 4 of RELN, has been successfully replicated in subsequent investigations, implying its potential contribution to schizophrenia susceptibility. To investigate the association of rs7341475 with schizophrenia in Chinese populations, a case-control association analysis was conducted with samples from Yuxi (400 cases and 400 controls) in southwestern China. The results do not indicate any association of rs7341475 with schizophrenia, which suggests it is not a risk SNP for schizophrenia in Han Chinese.

Association of RELN promoter SNPs with schizophrenia in the Chinese population.

Previous research on gene expression analysis and association tests have suggested that RELN is a risk gene for schizophrenia in world populations. Based on the reported down-regulation of RELN in schizophrenia patients compared with normal subjects, we speculated that variants in the RELN promoter region may confer risk for schizophrenia. In this study, we investigated the associations of three SNPs in the promoter region of RELN with schizophrenia in a case-control sample from southwestern China (940 cases and 1 369 controls). The results suggested that none of the SNPs showed significant associations in our sample, indicating the risk variants for schizophrenia in RELN may not be located in the promoter region. We also performed meta-analysis by combining our data with previously reported data on the Chinese population with a total sample size of 2 843 individuals, and the result remained non-significant. Collectively, our results suggested variants in the RELN promoter may not harbor risk SNPs associated with schizophrenia in the Chinese population.

[Smoking prevention and control among students of middle schools in Shanghai].

OBJECTIVE: To appraise the effectiveness of a school-based smoking control program and explore the effective smoking prevention and control intervention model in middle schools. METHODS: Through two phase cluster sampling, students in grade 1 and grade 2 of two schools were assigned to intervention group and control group. Comprehensive smoking intervention was implemented in the intervention group for one year. The assessment was carried out through three phases: baseline, after intervention, and 6-month after intervention. RESULTS: After intervention, the proportion of attempt smoking rate in the recent one year decreased from 9.7% to 4.2% and the heavy passive smoking rate declined from 15.5% to 12.2%. In addition, some knowledge, attitudes and skills related to tobacco prevention among the students in the intervention group were significantly improved and most effects can be sustained at 6-month follow up survey. CONCLUSION: It is concluded that tobacco control in middle school was effective and feasible and it should be closely integrated with tobacco control in the community.

[Smoking prevention and control among elementary school students in Xuhui district, Shanghai].

OBJECTIVE: To evaluate the effectiveness of a school-based smoking prevention and control intervention program among elementary school students. METHODS: Through two phase cluster sampling, 566 pupils in grade 4 and grade 5 of two schools were assigned to intervention group and control group. One year comprehensive smoking intervention was conducted in the intervention group. The assessment was carried out through three questionnaires: pre- and post-intervention, 6-month after intervention. RESULTS: After one year intervention, pupils in the intervention group significantly improved their knowledge and attitudes related to tobacco use. The rate of attempting smoking decreased form 7.8% to 2.6% and the rate of passive smoking from 53.6% to 41.8%. The difference between the intervention and control groups was statistically significant. However, several index started to decline at 6-month follow up survey. CONCLUSION: The result demonstrated the effectiveness and feasibility of tobacco control in elementary school and the positive effect must be developed.