Yanhua Su

The Neuronal Channel NALCN Contributes Resting Sodium Permeability and Is Required for Normal Respiratory Rhythm

Sodium plays a key role in determining the basal excitability of the nervous systems through the resting leak Na(+) permeabilities, but the molecular identities of the TTX- and Cs(+)-resistant Na(+) leak conductance are totally unknown. Here we show that this conductance is formed by the protein NALCN, a substantially uncharacterized member of the sodium/calcium channel family. Unlike any of the other 20 family members, NALCN forms a voltage-independent, nonselective cation channel. NALCN mutant mice have a severely disrupted respiratory rhythm and die within 24 hours of birth. Brain stem-spinal cord recordings reveal reduced neuronal firing. The TTX- and Cs(+)-resistant background Na(+) leak current is absent in the mutant hippocampal neurons. The resting membrane potentials of the mutant neurons are relatively insensitive to changes in extracellular Na(+) concentration. Thus, NALCN, a nonselective cation channel, forms the background Na(+) leak conductance and controls neuronal excitability.

Molecular and Functional Characterization of a Family of Amino Acid Transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.

Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80

Several neurotransmitters act through G-protein-coupled receptors to evoke a ‘slow’ excitation of neurons. These include peptides, such as substance P and neurotensin, as well as acetylcholine and noradrenaline. Unlike the fast (approximately millisecond) ionotropic actions of small-molecule neurotransmitters, the slow excitation is not well understood at the molecular level, but can be mainly attributed to suppressing K+ currents and/or activating a non-selective cation channel. The molecular identity of this cation channel has yet to be determined; similarly, how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. Here we show that, in the mouse hippocampal and ventral tegmental area neurons, substance P and neurotensin activate a channel complex containing NALCN and a large previously unknown protein UNC-80. The activation by substance P through TACR1 (a G-protein-coupled receptor for substance P) occurs by means of a unique mechanism: it does not require G-protein activation but is dependent on Src family kinases. These findings identify NALCN as the cation channel activated by substance P receptor, and suggest that UNC-80 and Src family kinases, rather than a G protein, are involved in the coupling from receptor to channel.

Shoot‐supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis

Deposition of ammonium (NH₄+) from the atmosphere is a substantial environmental problem. While toxicity resulting from root exposure to NH₄+ is well studied, little is known about how shoot-supplied ammonium (SSA) affects root growth. In this study, we show that SSA significantly affects lateral root (LR) development. We show that SSA inhibits lateral root primordium (LRP) emergence, but not LRP initiation, resulting in significantly impaired LR number. We show that the inhibition is independent of abscisic acid (ABA) signalling and sucrose uptake in shoots but relates to the auxin response in roots. Expression analyses of an auxin-responsive reporter, DR5:GUS, and direct assays of auxin transport demonstrated that SSA inhibits root acropetal (rootward) auxin transport while not affecting basipetal (shootward) transport or auxin sensitivity of root cells. Mutant analyses indicated that the auxin influx carrier AUX1, but not the auxin efflux carriers PIN-FORMED (PIN)1 or PIN2, is required for this inhibition of LRP emergence and the observed auxin response. We found that AUX1 expression was modulated by SSA in vascular tissues rather than LR cap cells in roots. Taken together, our results suggest that SSA inhibits LRP emergence in Arabidopsis by interfering with AUX1-dependent auxin transport from shoot to root.

Regulation by External K+ in a Maize Inward Shaker Channel Targets Transport Activity in the High Concentration Range

An inward Shaker K+ channel identified in Zea mays (maize), ZmK2.1, displays strong regulation by external K+ when expressed in Xenopus laevis (African clawed frog) oocytes or COS cells. ZmK2.1 is specifically activated by K+ with an apparent Km close to 15 mM independent of the membrane hyperpolarization level. In the absence of K+, ZmK2.1 appears to enter a nonconducting state. Thus, whatever the membrane potential, this maize channel cannot mediate K+ influx in the submillimolar concentration range, unlike its relatives in Arabidopsis thaliana. Its expression is restricted to the shoots, the strongest signal (RT-PCR) being associated with vascular/bundle sheath strands. Based on sequence and gene structure, the closest relatives of ZmK2.1 in Arabidopsis are K+ Arabidopsis Transporter 1 (KAT1) (expressed in guard cells) and KAT2 (expressed in guard cells and leaf phloem). Patch-clamp analyses of guard cell protoplasts reveal a higher functional diversity of K+ channels in maize than in Arabidopsis. Channels endowed with regulation by external K+ similar to that of ZmK2.1 (channel activity regulated by external K+ with a Km close to 15 mM, regulation independent of external Ca2+) constitute a major component of the maize guard cell inward K+ channel population. The presence of such channels in maize might reflect physiological traits of C4 and/or monocotyledonous plants.

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.

The differing responses of two Arabidopsis ecotypes to ammonium are modulated by the photoperiod regime

Responses to excessive ammonium (NH4+) were compared between two Arabidopsis ecotypes (Col-0, JA22) with respect to different photoperiods in hydroponics. In this study, we showed that external extra NH4+ led to severe growth suppression, accumulations of free NH4+ and amino acids and increased the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in shoots of the two Arabidopsis ecotypes. However, the levels of free NH4+ and total amino acids increased, whereas the activities of GS, NADH-dependent glutamate synthase and GDH decreased under the continuous light when compared with the light (16xa0h)–dark (8xa0h) cycle photoperiod. Statistical analyses suggested that strong correlations exist among the growth reduction, accumulations of free NH4+, total amino acids and levels of GS activity in shoots under the high NH4+ stress regardless of the photoperiod regimes. Interestingly, under the continuous light, Col-0 showed more resistant to such growth reduction and maintained about onefold higher capability of converting excess free NH4+ into amino acids, with onefold higher GS activity induced by the external NH4+ when compared with JA22. In contrast, these differences were abolished between Col-0 and JA22 under the light–dark cycle condition. Taken together, our results conclude that the sensitivity to NH4+ of Col-0 and JA22 is changed between the continuous light and the light–dark cycle photoperiod, which is correlative to the alteration of the GS activity in shoots.

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

Key messageFunctional identification of a relatively lower affinity ammonium transporter, OsAMT1;1, which is a proton-independent feedback regulated ammonium transporter in rice.AbstractRice 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+ (110–129xa0μM 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.

Over-expression of ApKUP3 enhances potassium nutrition and drought tolerance in transgenic 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.

Cs-miR156 is involved in the nitrogen form regulation of catechins accumulation in tea plant (Camellia sinensis L.).

The nitrogen source affects the growth of tea plants and regulates the accumulation of catechins in the leaves. In this report, we assessed the influences of NH4(+) and NO3(-) on plant growth, catechins accumulation and associated gene expression. Compared with the preferential nitrogen source NH4(+), when NO3(-) was supplied as the sole nitrogen source, tea plants showed similar symptoms with the nitrogen-free treatments and showed lower nitrogen, free amino acid accumulation, chlorophyll content and biomass gain, indicating NO3(-) was not efficiently used by these plants. However, the total shoot catechins content was significantly higher for NO3(-) treatments than that for NH4(+) treatment or combined NH4(+)+NO3(-) treatment, suggesting that, in addition to its influence on plant growth, the nitrogen form regulated the accumulation of catechins in tea. The expression of catechins biosynthesis-related genes was associated with the regulation of catechins accumulation and composition changes mediated by nitrogen form. PAL, CHS, CHI, and DFR genes exhibited higher expression levels in plants supplied with NO3(-), in which the transcript level of DFR in the shoots was significantly correlated with the catechins content. In the end, we identified a new function for the Cs-miR156, which was drastically induced through NH4(+). Moreover, a potential mechanism of the Cs-miR156 pathway in regulating catechins biosynthesis in tea plants has been suggested, with particular respect to nitrogen forms. Cs-miR156 might repress the expression of the target gene SPL to regulate the DFR gene, which plays a vital role in catechins biosynthesis.