Stimulation of nodulation in Medicago truncatula by low concentrations of ammonium: quantitative reverse transcription PCR analysis of selected genes.

Although mineral nitrogen generally has negative effects on nodulation in legume-rhizobia symbioses, low concentrations of ammonium stimulate nodulation in some legumes. In this study, the effects of ammonium and nitrate on growth, nodulation and expression of 2 nitrogen transport and 12 putative nodulation-related genes of the model symbiosis of Medicago truncatula - Sinorhizobium meliloti are investigated. After 3 weeks of hydroponic growth, whole-plant nodulation was enhanced in all the ammonium treatments and up to three-fold in the 0.5 mM treatment compared with the zero-nitrogen control. Specific nodulation (nodules g(-1) root dry weight) was greatly stimulated in the 0.1 and 0.5 mM NH4+ treatments, to a lower extent in the 0.1 mM NO3- treatment, and inhibited in all other treatments. Expression of the 14 selected genes was observed at 0, 6, 12 and 24 h after exposure to rhizobia and nitrogen. Expression of nitrogen transporter genes increased significantly, but responses of the three genes putatively associated with symbiosis signaling/nodule initiation were mixed. There were infrequent responses of genes coding for an ABA-activated protein kinase or a gibberellin-regulated protein, but an ethylene-responsive element-binding factor showed increased expression in various treatments and sampling times. Three auxin-responsive genes and three cytokinin-responsive genes showed varied responses to ammonium and nitrate. This study indicates that low concentrations of ammonium stimulate nodulation in M. truncatula, but the data were inconclusive in verifying the hypothesis that a relatively high ratio of cytokinin to auxin in roots may be an underlying mechanism in this stimulation of nodulation.

[1]  A. Cutler,et al.  Gene expression during seed maturation in Brassica napus in relation to the induction of secondary dormancy. , 2007, Genomics.

[2]  J. B. Reid,et al.  Defective Long-Distance Auxin Transport Regulation in the Medicago truncatula super numeric nodules Mutant1[W] , 2006, Plant Physiology.

[3]  P. Tillard,et al.  A Central Role for the Nitrate Transporter NRT2.1 in the Integrated Morphological and Physiological Responses of the Root System to Nitrogen Limitation in Arabidopsis1 , 2006, Plant Physiology.

[4]  S. Lievens,et al.  Gibberellins Are Involved in Nodulation of Sesbania rostrata1 , 2005, Plant Physiology.

[5]  F. Daniel-Vedele,et al.  The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. M. Williams,et al.  Effect of gibberellins and the growth retardant CCC on the nodulation of soya , 1984, Plant and Soil.

[7]  Jiannan Guo.  The differential effects of nitrate and ammonium on the growth, nodulation and nitrogen fixation of Medicago truncatula Gaertn. and the analysis of differential gene expression during the initiation of symbiosis with Sinorhizobium meliloti , 2005 .

[8]  J. Frugoli,et al.  The PIN and LAX families of auxin transport genes in Medicago truncatula , 2004, Molecular Genetics and Genomics.

[9]  J. Vessey,et al.  Further investigation of the roles of auxin and cytokinin in the NH4+‐induced stimulation of nodulation using white clover transformed with the auxin‐sensitive reporter GH3:gusA , 2004 .

[10]  R. Meeley,et al.  Expression of the maize GALACTINOL SYNTHASE gene family: (II) Kernel abscission, environmental stress and myo‐inositol influences accumulation of transcript in developing seeds and callus cells , 2004 .

[11]  S. Yoshida,et al.  Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species. , 2004, Plant & cell physiology.

[12]  M. Timko,et al.  Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.) , 2003, Plant and Soil.

[13]  E. Liscum,et al.  Genetics of Aux/IAA and ARF action in plant growth and development , 2002, Plant Molecular Biology.

[14]  J. Vessey,et al.  The stimulating effect of ammonium on nodulation in Pisum sativum L. is not long lived once ammonium supply is discontinued , 1997, Plant and Soil.

[15]  Y. Kapulnik,et al.  The role of phytohormones in plant-microbe symbioses , 1997, Plant and Soil.

[16]  A. Hirsch,et al.  Plant hormones and nodulation: what's the connection? , 1994, Plant Molecular Biology.

[17]  A. Hirsch,et al.  Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips , 1994, Plant Molecular Biology.

[18]  J. E. Harper,et al.  Effect of abscisic acid application on root isoflavonoid concentration and nodulation of wild-type and nodulation-mutant soybean plants , 1993, Plant and Soil.

[19]  E. Beck,et al.  Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status , 1993, Planta.

[20]  M. L. G. Ramos,et al.  Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.) , 1993, Plant and Soil.

[21]  F. Below,et al.  Role of cytokinin in enhanced productivity of maize supplied with NH4+ and NO3− , 1992, Plant and Soil.

[22]  R. Dixon,et al.  Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis , 1991, Plant Molecular Biology.

[23]  P. Gallusci,et al.  Synchronous expression of leghaemoglobin genes inMedicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate , 1991, Plant Molecular Biology.

[24]  B. Lugtenberg,et al.  The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype , 1989, Planta.

[25]  Tatsuo Kakimoto,et al.  Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate. , 2004, The Plant journal : for cell and molecular biology.

[26]  H. van Keulen,et al.  Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean , 2004, Plant and Soil.

[27]  J. Vessey,et al.  Low concentrations of ammonium inhibit specific nodulation (nodule number g-1 root DW) in soybean (Glycine max [L.] Merr.) , 2004, Plant and Soil.

[28]  U. Mathesius,et al.  Signaling Interactions During Nodule Development , 2003, Journal of Plant Growth Regulation.

[29]  J. Vessey,et al.  Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum , 2003 .

[30]  M. Okamoto,et al.  The regulation of nitrate and ammonium transport systems in plants. , 2002, Journal of experimental botany.

[31]  R. Luciński,et al.  Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes. , 2002, Acta biochimica Polonica.

[32]  G. Oldroyd,et al.  Ethylene Inhibits the Nod Factor Signal Transduction Pathway of Medicago truncatula , 2001, The Plant Cell Online.

[33]  T. Kakimoto.  Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases. , 2001, Plant & cell physiology.

[34]  Marie-Agathe Lorteau,et al.  Effects of cytokinin on ethylene production and nodulation in pea (Pisum sativum) cv. Sparkle. , 2001, Physiologia plantarum.

[35]  S. May,et al.  Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development. , 2001, Molecular plant-microbe interactions : MPMI.

[36]  U. Mathesius.  Flavonoids induced in cells undergoing nodule organogenesis in white clover are regulators of auxin breakdown by peroxidase. , 2001, Journal of experimental botany.

[37]  M. Bennett,et al.  Auxin transport: providing a sense of direction during plant development. , 2000, Biochemical Society transactions.

[38]  K. Minamisawa,et al.  Effects of ethylene precursor and inhibitors for ethylene biosynthesis and perception on nodulation in Lotus japonicus and Macroptilium atropurpureum. , 2000, Plant & cell physiology.

[39]  M. Crespi,et al.  Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition. , 2000, Molecular plant-microbe interactions : MPMI.

[40]  S. Howitt,et al.  Structure, function and regulation of ammonium transporters in plants. , 2000, Biochimica et biophysica acta.

[41]  J. Schjoerring,et al.  Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley. , 2000, Plant physiology.

[42]  W. Frommer,et al.  Three Functional Transporters for Constitutive, Diurnally Regulated, and Starvation-Induced Uptake of Ammonium into Arabidopsis Roots , 1999, Plant Cell.

[43]  P. Gresshoff,et al.  Nitrate- and inoculation-enhanced ethylene biosynthesis in soybean roots as a possible mediator of nodulation control , 1999 .

[44]  T. Guilfoyle.  Aux/IAA proteins and auxin signal transduction , 1998 .

[45]  H. Spaink,et al.  Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides. , 1998, The Plant journal : for cell and molecular biology.

[46]  F. Ligero,et al.  Nitrate‐induced ethylene biosynthesis and the control of nodulation in alfalfa , 1998 .

[47]  B. Sotta,et al.  Effects of NO3−, NH4+ and urea nutrition on endogenous levels of IAA and four cytokinins in two epiphytic bromeliads , 1997 .

[48]  D. Cook,et al.  A Legume Ethylene-Insensitive Mutant Hyperinfected by Its Rhizobial Symbiont , 1997, Science.

[49]  J. Weinman,et al.  Chalcone Synthase Gene Expression in Transgenic Subterranean Clover Correlates with Localised Accumulation of Flavonoids , 1997 .

[50]  B. W. Shirley.  Flavonoid biosynthesis: ‘new’ functions for an ‘old’ pathway , 1996 .

[51]  S. Long.  Rhizobium symbiosis: nod factors in perspective. , 1996, The Plant cell.

[52]  M. Crespi,et al.  Nod factors and cytokinins induce similar cortical cell division, amyloplast deposition and MsEnod12A expression patterns in alfalfa roots , 1996 .

[53]  O. Junttila,et al.  Differential rates of inhibition of N2 fixation by sustained low concentrations of NH4+ and NO3- in northern ecotypes of white clover (Trifolium repens L.) , 1996 .

[54]  R. Dickstein,et al.  The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover , 1996, Plant physiology.

[55]  J. Dénarié,et al.  Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. , 1996, Annual review of biochemistry.

[56]  N. Suganuma,et al.  Enhanced production of ethylene by soybean roots after inoculation with Bradyrhizobium japonicum , 1995 .

[57]  A. Kondorosi,et al.  Differential expression of cDNA coding for chalcone reductase, a key enzyme of the 5-deoxyflavonoid pathway, under various stress conditions in Medicago sativa , 1995 .

[58]  R. Dixon,et al.  Stress-Induced Phenylpropanoid Metabolism. , 1995, The Plant cell.

[59]  A. Kondorosi,et al.  Alfalfa Root Flavonoid Production Is Nitrogen Regulated , 1995, Plant physiology.

[60]  J. Kijne,et al.  Ethylene prevents nodulation of Vicia sativa ssp. nigra by exopolysaccharide-deficient mutants of Rhizobium leguminosarum bv. viciae , 1995 .

[61]  E. Kondorosi,et al.  Cell and Molecular Biology of Rhizobium-Plant , 1994 .

[62]  P. Wojtaszek,et al.  Role of Nitrogen and Plant Growth Regulators in the Exudation and Accumulation of lsoflavonoids by Roots of Intact White (Lupin (Lupinus albus L.) Plants , 1993 .

[63]  R. Dixon,et al.  Molecular characterization and expression of alfalfa isoliquiritigenin 2'-O-methyltransferase, an enzyme specifically involved in the biosynthesis of an inducer of Rhizobium meliloti nodulation genes. , 1993, The Plant journal : for cell and molecular biology.

[64]  J. Vessey,et al.  Effect of low static nitrate concentrations on mineral nitrogen uptake, nodulation, and nitrogen fixation in field pea , 1993 .

[65]  J. Vessey,et al.  Nodulation response of autoregulated or NH4+‐inhibited pea (Pisum sativum) after transfer to stimulatory (low) concentrations of NH4+ , 1993 .

[66]  W. Hunter.  Ethylene Production by Root Nodules and Effect of Ethylene on Nodulation in Glycine max , 1993, Applied and environmental microbiology.

[67]  C. Larsson,et al.  Nitrate regulation of zeation riboside levels in barley roots: effects of inhibitors of N assimilation and comparison with ammonium , 1993 .

[68]  T. Bisseling,et al.  The PsENOD12 Gene Is Expressed at Two Different Sites in Afghanistan Pea Pseudonodules Induced by Auxin Transport Inhibitors. , 1992, Plant physiology.

[69]  T. Larue,et al.  Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle. , 1992, Plant physiology.

[70]  R. Graham,et al.  Effect of four nitrogen compounds on nodulation and nitrogen fixation in Faba bean, white lupin and medic plants , 1992 .

[71]  C. Dehio,et al.  The early nodulin gene SrEnod.2 from Sesbania is inducible by cytokinin , 1992 .

[72]  C. Raper,et al.  Stimulation of nodulation in field peas (Pisum sativum) by low concentrations of ammonium in hydroponic culture. , 1992, Physiologia plantarum.

[73]  C. Larsson,et al.  Nitrate-regulated growth and cytokinin responses in seminal roots of barley. , 1992, Plant physiology.

[74]  J. E. Harper,et al.  Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants. , 1991, Plant physiology.

[75]  G. B. Kerbauy,et al.  Effects of Nitrogen Source on Growth Rates and Levels of Endogenous Cytokinins and Chlorophyll in Protocorms of Epidendrum fulgens , 1991 .

[76]  T. Larue,et al.  Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle. , 1991, Plant physiology.

[77]  R. Dixon,et al.  Stress responses in alfalfa (Medicago sativa L.) VII. Induction of defence related mRNAs in elicitor-treated cell suspension cultures , 1990 .

[78]  R. Dixon,et al.  Activation, structure, and organization of genes involved in microbial defense in plants. , 1990, Advances in genetics.

[79]  C M Joseph,et al.  A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti. , 1989, Plant physiology.

[80]  T. Bisseling,et al.  Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[81]  D. Scheel,et al.  Physiology and Molecular Biology of Phenylpropanoid Metabolism , 1989 .

[82]  P. Kuiper,et al.  Effects of internal and external cytokinin concentrations on root growth and shoot to root ratio of Plantago major ssp Pleiosperma at different nutrient conditions , 1988 .

[83]  P. Rubery,et al.  Naturally Occurring Auxin Transport Regulators , 1988, Science.

[84]  P. Wong,et al.  Inhibition of legume nodule formation and N2 fixation by nitrate , 1988 .

[85]  J. Olivares,et al.  Evolution of Ethylene from Roots of Medicago sativa Plants Inoculated with Rhizobium meliloti , 1986 .

[86]  J. Torrey.  Endogenous and exogenous influences on the regulation of lateral root formation , 1986 .

[87]  W. Wallace,et al.  Effects of Nitrate and Ammonium on Nitrogenase (C2H2 Reduction) Activity of Swards of Subterranean Clover, Trifolium subterraneum L. , 1986 .

[88]  M. Jackson.  New Root Formation in Plants and Cuttings , 1986, Developments in Plant and Soil Sciences.

[89]  G. Rys,et al.  Effect of Nitrogen Form and Counterion on Establishment of the Rhizobium trifolii-Trifolium repens Symbiosis , 1984 .

[90]  P. Wareing,et al.  Effects of Mineral Nutrition on Endogenous Cytokinins in Plants of Sunflower (Helianthus annuus L.) , 1979 .

[91]  J. Torrey,et al.  Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L. , 1976, Plant physiology.