Phosphorous accumulation associated with mitochondrial PHT3-mediated enhanced arsenate tolerance in Chlamydomonas reinhardtii.

Arsenate is a highly toxic element and excessive accumulation of arsenic in the aquatic environment easily triggers a problem threatening the ecological health. Phytoremediation has been widely explored as a method to alleviate As contamination. Here, the green algae, Chlamydomonas reinhardtii was investigated by profiling the accumulation of arsenate and phosphorus, which share the same uptake pathway, in response to arsenic stress. Both C. reinhardtii wild type C30 and the Crpht3 mutant were cultured under arsenic stress, and demonstrated a similar growth phenotype under limited phosphate supply. Sufficient phosphate obviously increased the uptake of polyphosphate and intercellular phosphate in the Crpht3 mutant, which increased the arsenic tolerance of the Crpht3 mutant under stress from 500 µmol L-1 As(V). Upregulation of the PHT3 gene in the Crpht3 mutant increased accumulation of phosphate in the cytoplasm under arsenate stress, which triggered a regulatory role for the differentially expressed genes that mediated improvement of the glutathione redox cycle, antioxidant activity and detoxification. While the wild type C30 showed weak arsenate tolerance because of little phosphate accumulation. These results suggest that the enhanced arsenic tolerance of the Crpht3 mutant is regulated by the PHT3 gene mediation. This study provides insight onto the responsive mechanisms of the PHT3 gene-mediated in alleviating arsenic toxicity in plants.

Molecular identification and physiological functional analysis of NtNRT1.1B that mediated nitrate long-distance transport and improved plant growth when overexpressed in tobacco.

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( NO 3 - ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for NO 3 - , little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating NO 3 - transport associated with tobacco growth on NO 3 - nutrition. Heterologous expression of NtNRT1.1B in the NO 3 - uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM NO 3 - , suggesting a possible molecular function of NtNRT1.1B in the import of NO 3 - into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of NO 3 - permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including NO 3 - , after 3 days' N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, NO 3 - , and even NH 4 + in plants supplied with NO 3 - ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- NO 3 - root influx assays, which showed 15%-20% higher NO 3 - deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for NO 3 - at a K m of around 30-45 µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher NO 3 - in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.

Genome-wide identification and expression analysis of DREB genes in alfalfa (Medicago sativa) in response to cold stress.

Dehydration-responsive element-binding proteins (DREBs) belong to members of the AP2/ERF transcription factor superfamily, which has been reported to involve various abiotic-stress responses and tolerance in plants. However, research on the DREB-family is still limited in alfalfa (Medicago sativa L.), a forage legume cultivated worldwide. The recent genome-sequence release of the alfalfa cultivar "XinJiangDaYe" allowed us to identify 172 DREBs by a multi-step homolog search. The phylogenetic analysis indicated that such MsDREBs could be classified into 5 groups, namely A-1 (56 members), A-2 (39), A-3 (3), A-4 (61) and 13 (A-5 (13), thus adding substantial new members to the DREB-family in alfalfa. Furthermore, a comprehensive survey in silico of conserved motif, gene structure, molecular weight, and isoelectric point (pI) as well as gene expression was conducted. The resulting data showed that, for cold-stress response, 33 differentially expressed MsDREBs were identified with a threshold of Log2-fold > 1, and most of which were transcriptionally upregulated within 48 h during a cold treatment(s). Moreover, the expression profiling of MsDREBs from two ecotypes of alfalfa subspecies i.e. M. sativa ssp. falcata (F56, from a colder region of Central Asia) and M. sativa ssp. sativa (B47, from Near East) revealed that most of the cold-stress responsive MsDREBs exhibited a significantly lower expression in F56, leading to a proposal of the existence of a distinct mechanism(s) for cold tolerance regulated by DREB-related action, which would have been evolved in alfalfa with a genotypic specificity. Additionally, by examining the transcriptome of a freezing-tolerance species (M. sativa cv. Zhaodong), eight DREBs were found to be implicated in a long-term freezing-stress adaptation with a great potential. Taken together, the current genome-wide identification in alfalfa points to the importance of some MsDREBs in the cold-stress response, providing some promising molecular targets to be functionally characterized for the improvement of cold tolerance in crops including alfalfa.

Polyamine: A Potent Ameliorator for Plant Growth Response and Adaption to Abiotic Stresses Particularly the Ammonium Stress Antagonized by Urea.

Polyamine(s) (PA, PAs), a sort of N-containing and polycationic compound synthesized in almost all organisms, has been recently paid considerable attention due to its multifarious actions in the potent modulation of plant growth, development, and response to abiotic/biotic stresses. PAs in cells/tissues occur mainly in free or (non- or) conjugated forms by binding to various molecules including DNA/RNA, proteins, and (membrane-)phospholipids, thus regulating diverse molecular and cellular processes as shown mostly in animals. Although many studies have reported that an increase in internal PA may be beneficial to plant growth under abiotic conditions, leading to a suggestion of improving plant stress adaption by the elevation of endogenous PA via supply or molecular engineering of its biosynthesis, such achievements focus mainly on PA homeostasis/metabolism rather than PA-mediated molecular/cellular signaling cascades. In this study, to advance our understanding of PA biological actions important for plant stress acclimation, we gathered some significant research data to succinctly describe and discuss, in general, PA synthesis/catabolism, as well as PA as an internal ameliorator to regulate stress adaptions. Particularly, for the recently uncovered phenomenon of urea-antagonized NH4 +-stress, from a molecular and physiological perspective, we rationally proposed the possibility of the existence of PA-facilitated signal transduction pathways in plant tolerance to NH4 +-stress. This may be a more interesting issue for in-depth understanding of PA-involved growth acclimation to miscellaneous stresses in future studies.

Plant DNA methylation is sensitive to parent seed N content and influences the growth of rice.

BACKGROUND: Nitrogen (N) is an important nutrient for plant growth, development, and agricultural production. Nitrogen stress could induce epigenetic changes in plants. In our research, overexpression of the OsNAR2.1 line was used as a testing target in rice plants with high nitrogen-use efficiency to study the changes of rice methylation and growth in respond of the endogenous and external nitrogen stress. RESULTS: Our results showed that external N deficiency could decrease seed N content and plant growth of the overexpression line. During the filial growth, we found that the low parent seed nitrogen (LPSN) in the overexpression line could lead to a decrease in the filial seed nitrogen content, total plant nitrogen content, yield, and OsNAR2.1 expression (28, 35, 23, and 55%, respectively) compared with high parent seed nitrogen (HPSN) in high nitrogen external supply. However, such decreases were not observed in wild type. Furthermore, methylation sequencing results showed that LPSN caused massive gene methylation changes, which enriched in over 20 GO pathways in the filial overexpression line, and the expression of OsNAR2.1 in LPSN filial overexpression plants was significantly reduced compared to HPSN filial plants in high external N, which was not shown in wild type. CONCLUSIONS: We suggest that the parent seed nitrogen content decreased induced DNA methylation changes at the epigenetic level and significantly decreased the expression of OsNAR2.1, resulting in a heritable phenotype of N deficiency over two generations of the overexpression line.

Evidence that exogenous urea acts as a potent cue to alleviate ammonium-inhibition of root system growth of cotton plant (Gossypium hirsutum).

Many plants grown with low-millimolar concentration of NH4 + as a sole nitrogen source develop NH4 + -toxicity symptoms. To date, crucial molecular identities and a practical approach involved in the improvement of plant NH4 + -tolerance remain largely unknown. By phenotyping of upland cotton grown on varied nitrogen forms, we came across a phenomenon that caused sub-millimolar concentrations of urea (e.g., up 50 µM) to repress the growth inhibition of roots and whole plant cultivated in a NH4 + -containing nutrient solution. A growth-recovery assay revealed that the relief in NH4 + -inhibited growth required only a short-term exposure (≧12 h) of the roots to urea, implying that urea could elicit an internal signaling and be involved in antagonizing NH4 + -sensitivity. Intriguingly, split-root experiments demonstrated that low urea occurrence in one root-half could efficaciously stimulate not only supplied root but also the root-half grown in NH4 + -solution without urea, indicating the existence of urea-triggered local and systemic long-distance signaling. In the split-root experiment we also observed high arginase activity, strong arginine reduction and remarkable upregulation of polyamine biosynthesis-related genes (ADC1/2, SPDS and SPMS). Therefore, we suggest that external urea might serve as an effective cue (signal molecule) in an arginine-/polyamine-related process for ameliorating NH4 + -suppressed root growth, providing a novel aspect for deeper exploring and understanding plant NH4 + -tolerance.

Phenotypical evidence of effective amelioration of ammonium-inhibited plant (root) growth by exogenous low urea.

Ammonium and nitrate are major soil inorganic-nitrogen sources for plant growth, but many species cultivated with even low millimolar NH4+ as a sole N form display a growth retardation. To date, critical biological components and applicable approaches involved in the effective enhancement of NH4+ tolerance remain to be thoroughly explored. Here, we report phenotypical traits of urea-dependent improvement of NH4+-suppressed plant/root growth. Urea at 0.1 mM was sufficient to remarkably stimulate NH4+ (3 mM)-fed cotton growth, showing a 2.5∼4-fold increase in shoot- and root-biomass and total root-length, 20 % higher GS activity, 18 % less NH4+-accumulation in roots, and a comparable plant total-N content compared to the control, implying a novel role for urea in cotton NH4+detoxification. A similar phenomenon was observed in tobacco and rice. Moreover, comparisons between twelve NH4+-grown Arabidopsis accessions revealed a great degree of natural variation in their root-growth response to low urea, with WAR and Blh-1 exhibiting the most significant increase in primary- and lateral-root length and numbers, and Sav-0 and Edi-0 being the most insensitive. Such phenotypical evidence suggests a common ability of plants to accommodate NH4+-stress by responding to exogenous urea, providing a novel aspect for further understanding the process of urea-dependent plant NH4+ tolerance.

Mutagenesis Reveals That the OsPPa6 Gene Is Required for Enhancing the Alkaline Tolerance in Rice.

Alkaline stress (AS) is one of the abiotic stressful factors limiting plant's growth and development. Inorganic pyrophosphatase is usually involved in a variety of biological processes in plant in response to the abiotic stresses. Here, to clarify the responsive regulation of inorganic pyrophosphatase in rice under AS, the mutagenesis of the OsPPa6 gene encoding an inorganic pyrophosphatase in rice cv. Kitaake (Oryza sativa L. ssp. japonica) was performed by the CRISPR/Cas9 system. Two homozygous independent mutants with cas9-free were obtained by continuously screening. qPCR reveals that the OsPPa6 gene was significantly induced by AS, and the mutagenesis of the OsPPa6 gene apparently delayed rice's growth and development, especially under AS. Measurements demonstrate that the contents of pyrophosphate in the mutants were higher than those in the wild type under AS, however, the accumulation of inorganic phosphate, ATP, chlorophyll, sucrose, and starch in the mutants were decreased significantly, and the mutagenesis of the OsPPa6 gene remarkably lowered the net photosynthetic rate of rice mutants, thus reducing the contents of soluble sugar and proline, but remarkably increasing MDA, osmotic potentials and Na+/K+ ratio in the mutants under AS. Metabonomics measurement shows that the mutants obviously down-regulated the accumulation of phosphorylcholine, choline, anthranilic acid, apigenin, coniferol and dodecanoic acid, but up-regulated the accumulation of L-valine, alpha-ketoglutarate, phenylpyruvate and L-phenylalanine under AS. This study suggests that the OsPPa6 gene is an important osmotic regulatory factor in rice, and the gene-editing of CRISPR/Cas9-guided is an effective method evaluating the responsive regulation of the stress-induced gene, and simultaneously provides a scientific support for the application of the gene encoding a soluble inorganic pyrophosphatase in molecular breeding.

Molecular identification of a root apical cell-specific and stress-responsive enhancer from an Arabidopsis enhancer trap line.

BACKGROUND: Plant root apex is the major part to direct the root growth and development by responding to various signals/cues from internal and soil environments. To study and understand root system biology particularly at a molecular and cellular level, an Arabidopsis T-DNA insertional enhancer trap line J3411 expressing reporters (GFP) only in the root tip was adopted in this study to isolate a DNA fragment. RESULTS: Using nested PCR, DNA sequencing and sequence homology search, the T-DNA insertion site(s) and its flanking genes were characterised in J3411 line. Subsequently, a 2000 bp plant DNA-fragment (Ertip1) upstream of the insert position of the coding T-DNA was in silico analysed, revealing certain putative promoter/enhancer cis-regulatory elements. Cloning and transformation of this DNA fragment and its truncated segments tagged with or without 35S minimal promoter (35Smini), all of which were fused with a GFP or GUS reporter, allowed to detect GFP and GUS expression mediated only by Ertip1 + 35mini (PErtip1+35Smini) specifically in the Arabidopsis root tip region. The PErtip1+35Smini activity was further tested to be strong and stable under many different growth conditions but suppressed by cold, salt, alkaline pH and higher ammonium and phosphorus. CONCLUSION: This work describes a promising strategy to isolate a tissue-/cell-specific enhancer sequence from the enhancer trap lines, which are publically available. The reported synthetic promoter i.e. PErtip1+35Smini may provide a valuable and potent molecular-tool for comprehensive investigation of a gene function related to root growth and development as well as molecular engineering of root-architectural formation aiming to improve plant growth.

Molecular identification and functional characterization of GhAMT1.3 in ammonium transport with a high affinity from cotton (Gossypium hirsutum L.).

Ammonium (NH4 + ) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH4 + movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH4 + uptake and molecular identification of certain NH4 + transporters (AMTs) from cotton (Gossypium hirustum). A substrate-influx assay using 15 N-isotope revealed that cotton possessed a high-affinity transport system with a Km of 58 µM for NH4 + . Sequence analysis showed that GhAMT1.1-1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1-1.3 expression mediated NH4 + permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko-mutant cells, allowing a growth restoration of both mutants on NH4 + . Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up-regulated by N-starvation, repressed by NH4 + resupply and regulated diurnally and age-dependently, suggesting that GhAMT1.3 should be a N-responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH4 + and enhanced total nitrogen accumulation (∼50% more), conforming with the observation of 2-fold more NH4 + absorption by GhAMT1.3-transformed qko plant roots during a 1-h root influx period. Together with its targeting to the PM and saturated transport kinetics with a Km of 72 µM for NH4 + , GhAMT1.3 is suggested to be a high-affinity NH4 + permease that may play a significant role in cotton NH4 + acquisition and utilization, adding a new member in the plant AMT family.

Coding-Sequence Identification and Transcriptional Profiling of Nine AMTs and Four NRTs From Tobacco Revealed Their Differential Regulation by Developmental Stages, Nitrogen Nutrition, and Photoperiod.

Although many members encoding different ammonium- and nitrate-transporters (AMTs, NRTs) were identified and functionally characterized from several plant species, little is known about molecular components for [Formula: see text]- and [Formula: see text] acquisition/transport in tobacco, which is often used as a plant model for biological studies besides its agricultural and industrial interest. We reported here the first molecular identification in tobacco (Nicotiana tabacum) of nine AMTs and four NRTs, which are respectively divided into four (AMT1/2/3/4) and two (NRT1/2) clusters and whose functionalities were preliminarily evidenced by heterologous functional-complementation in yeast or Arabidopsis. Tissue-specific transcriptional profiling by qPCR revealed that NtAMT1.1/NRT1.1 mRNA occurred widely in leaves, flower organs and roots; only NtAMT1.1/1.3/2.1NRT1.2/2.2 were strongly transcribed in the aged leaves, implying their dominant roles in N-remobilization from source/senescent tissues. N-dependent expression analysis showed a marked upregulation of NtAMT1.1 in the roots by N-starvation and resupply with N including [Formula: see text], suggesting a predominant action of NtAMT1.1 in [Formula: see text] uptake/transport whenever required. The obvious leaf-expression of other NtAMTs e.g., AMT1.2 responsive to N indicates a major place, where they may play transport roles associated with plant N-status and ([Formula: see text]-)N movement within aerial-parts. The preferentially root-specific transcription of NtNRT1.1/1.2/2.1 responsive to N argues their importance for root [Formula: see text] uptake and even sensing in root systems. Moreover, of all NtAMTs/NRTs, only NtAMT1.1/NRT1.1/1.2 showed their root-expression alteration in a typical diurnal-oscillation pattern, reflecting likely their significant roles in root N-acquisition regulated by internal N-demand influenced by diurnal-dependent assimilation and translocation of carbohydrates from shoots. This suggestion could be supported at least in part by sucrose- and MSX-affected transcriptional-regulation of NtNRT1.1/1.2. Thus, present data provide valuable molecular bases for the existence of AMTs/NRTs in tobacco, promoting a deeper understanding of their biological functions.

Molecular identification of tobacco NtAMT1.3 that mediated ammonium root-influx with high affinity and improved plant growth on ammonium when overexpressed in Arabidopsis and tobacco.

Although biological functions of ammonium (NH4+) transporters (AMTs) have been intensively studied in many plant species, little is known about molecular bases responsible for NH4+ movement in tobacco. Here, we reported the identification and functional characterization of a putative NH4+ transporter NtAMT1.3 from tobacco (Nicotiana tabacum). Analysis in silico showed that NtAMT1.3 encoded an integral membrane protein containing 464 amino acid residues and exhibiting 10 predicted transmembrane α-helices. Heterologous functionality study demonstrated that NtAMT1.3 expression facilitated NH4+ entry across plasma membrane of NH4+-uptake defective yeast and Arabidopsis qko mutant, allowing a restored growth of both yeast and Arabidopsis mutant on low NH4+. qPCR assay revealed that NtAMT1.3 was expressed in both roots and leaves and significantly up-regulated by nitrogen starvation and resupply of its putative substrate NH4+ and even nitrate, suggesting that NtAMT1.3 should represent a nitrogen-responsive gene. Critically, constitutive overexpression of NtAMT1.3 in tobacco per se improved obviously the growth of transgenic plants on NH4+ and enhanced leaf nitrogen (15% more) accumulation, consistent with observation of 35% more NH4+ uptake by the roots of transgenic lines in 20min root-influx test. Together with data showing its plasma membrane localization and saturated transport nature with Km of about 50µM for NH4+, we suggest that NtAMT1.3 acts an active NH4+ transporter that plays a significant role in NH4+ acquisition and utilization in tobacco.

Analyzing serial cDNA libraries revealed reactive oxygen species and gibberellins signaling pathways in the salt response of Upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: By comparing series full-length cDNA libraries stressed and control, the dynamic process of salt stress response in Upland cotton was studied, and reactive oxygen species and gibberellins signaling pathways were proposed. The Upland cotton is the most important fiber plant with highly salt tolerance. However, the molecular mechanism underlying salt tolerance in domesticated cotton was unclear. Here, seven full-length cDNA libraries were constructed for seedling roots of Upland cotton 'Zhong G 5' at 0, 3, 12 and 48 h after the treatment of control or 150 mM NaCl stress. About 3300 colonies in each library were selected robotically for 5'-end pyrosequencing, resulting in 20,358 expressed sequence tags (ESTs) totally. And 8516 uniESTs were then assembled, including 2914 contigs and 5602 singletons, and explored for Gene Ontology (GO) function. GO comparison between serial stress libraries and control reflected the growth regulation, stimulus response, signal transduction and biology regulation processes were conducted dynamically in response to salt stress. MYB, MYB-related, WRKY, bHLH, GRAS and ERF families of transcription factors were significantly enriched in the early response. 65 differentially expressed genes (DEGs), mainly associated with reactive oxygen species (ROS) scavenging, gibberellins (GAs) metabolism, signal transduction, transcription regulation, stress response and transmembrane transport, were identified and confirmed by quantitative real-time PCR. Overexpression of selected DEGs increased tolerance against salt stress in transgenic yeast. Results in this study supported that a ROS-GAs interacting signaling pathway of salt stress response was activated in Upland cotton. Our results provided valuable gene resources for further investigation of the molecular mechanism of salinity tolerance.

Molecular identification and functional analysis of a maize (Zea mays) DUR3 homolog that transports urea with high affinity.

MAIN CONCLUSION: Successful molecular cloning and functional characterization of a high-affinity urea permease ZmDUR3 provide convincing evidence of ZmDUR3 roles in root urea acquisition and internal urea-N-remobilization of maize plants. Urea occurs ubiquitously in both soils and plants. Being a major form of nitrogen fertilizer, large applications of urea assist cereals in approaching their genetic yield potential, but due to the low nitrogen-use efficiency of crops, this practice poses a severe threat to the environment through their hypertrophication. To date, except for paddy rice, little is known about the biological basis for urea movement in dryland crops. Here, we report the molecular and physiological characterization of a maize urea transporter, ZmDUR3. We show using gene prediction, PCR-based cloning and yeast complementation, that a functional full-length cDNA encoding a 731 amino acids-containing protein with putative 15 transmembrane α-helixes for ZmDUR3 was successfully cloned. Root-influx studies using (15)N-urea demonstrated ZmDUR3 catalyzes urea transport with a K m at ~9 µM when expressed in the Arabidopsis dur3-mutant. qPCR analysis revealed that ZmDUR3 mRNA in roots was significantly upregulated by nitrogen depletion and repressed by reprovision of nitrogen after nitrogen starvation, indicating that ZmDUR3 is a nitrogen-responsive gene and relevant to plant nitrogen nutrition. Moreover, detection of higher urea levels in senescent leaves and obvious occurrence of ZmDUR3 transcripts in phloem-cells of mature/aged leaves strongly implies a role for ZmDUR3 in urea vascular loading. Significantly, expression of ZmDUR3 complemented atdur3-mutant of Arabidopsis, improving plant growth on low urea and increasing urea acquisition. As it also targets to the plasma membrane, our data suggest that ZmDUR3 functions as an active urea permease playing physiological roles in effective urea uptake and nitrogen remobilization in maize.

A plant homolog of animal glutamate receptors is an ion channel gated by multiple hydrophobic amino acids.

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants have been implicated in root development, ion transport, and several metabolic and signaling pathways. AtGLR3.4, a plant iGluR homolog from Arabidopsis thaliana, has ion channel activity and is gated by asparagine, serine, and glycine. Using heterologous expression in Xenopus oocytes, we found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca(2+)-permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs. Seven of the 20 standard amino acids--mainly hydrophobic ones--acted as agonists, with methionine being most effective and most potent. Nine amino acids were antagonists, and four, including glutamate and glycine, had no effect on channel activity. We constructed a model of this previously uncharacterized ligand specificity and used knockout mutants to show that AtGLR1.4 accounts for methionine-induced membrane depolarization in Arabidopsis leaves.

Rice DUR3 mediates high-affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis.

• Despite the great agricultural and ecological importance of efficient use of urea-containing nitrogen fertilizers by crops, molecular and physiological identities of urea transport in higher plants have been investigated only in Arabidopsis. • We performed short-time urea-influx assays which have identified a low-affinity and high-affinity (K(m) of 7.55 µM) transport system for urea-uptake by rice roots (Oryza sativa). • A high-affinity urea transporter OsDUR3 from rice was functionally characterized here for the first time among crops. OsDUR3 encodes an integral membrane-protein with 721 amino acid residues and 15 predicted transmembrane domains. Heterologous expression demonstrated that OsDUR3 restored yeast dur3-mutant growth on urea and facilitated urea import with a K(m) of c. 10 µM in Xenopus oocytes. • Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis revealed upregulation of OsDUR3 in rice roots under nitrogen-deficiency and urea-resupply after nitrogen-starvation. Importantly, overexpression of OsDUR3 complemented the Arabidopsis atdur3-1 mutant, improving growth on low urea and increasing root urea-uptake markedly. Together with its plasma membrane localization detected by green fluorescent protein (GFP)-tagging and with findings that disruption of OsDUR3 by T-DNA reduces rice growth on urea and urea uptake, we suggest that OsDUR3 is an active urea transporter that plays a significant role in effective urea acquisition and utilisation in rice.

Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine.

Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.

Identification and characterization of proteins involved in rice urea and arginine catabolism.

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana.

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.

Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis.

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.