On the relationship between C and N fixation and amino acid synthesis in nodulated alfalfa (Medicago sativa)
Gemma Molero A B F , Guillaume Tcherkez C D , Jose Luis Araus B , Salvador Nogués B and Iker Aranjuelo B E
+ Author Affiliations
- Author Affiliations
A International Maize and Wheat Improvement Center (CIMMYT), El Batán, Texcoco, CP 56130, Mexico.
B Unitat de Fisologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain.
C Plateforme Métabolisme-Métabolome, IFR 87, Bât. 630, Université Paris Sud, 91405 Orsay cedex, France.
D Institut Universitaire de France, 103 Boulevard Saint Michel, 75005 Paris cedex, France.
E Instituto de Agrobiotecnología (IdAB), Universidad Pública de Navarra-CSIC-Gobierno de Navarra, Campus de Arrosadía, E-31192-Mutilva Baja, Spain.
F Corresponding author. Email: g.molero@cgiar.org
Functional Plant Biology 41(4) 331-341 https://doi.org/10.1071/FP13189
Submitted: 21 June 2013 Accepted: 31 October 2013 Published: 13 January 2014
Abstract
Legumes such as alfalfa (Medicago sativa L.) are vital N2-fixing crops accounting for a global N2 fixation of ~35 Mt N year–1. Although enzymatic and molecular mechanisms of nodule N2 fixation are now well documented, some uncertainty remains as to whether N2 fixation is strictly coupled with photosynthetic carbon fixation. That is, the metabolic origin and redistribution of carbon skeletons used to incorporate nitrogen are still relatively undefined. Here, we conducted isotopic labelling with both 15N2 and 13C-depleted CO2 on alfalfa plants grown under controlled conditions and took advantage of isotope ratio mass spectrometry to investigate the relationship between carbon and nitrogen turn-over in respired CO2, total organic matter and amino acids. Our results indicate that CO2 evolved by respiration had an isotopic composition similar to that in organic matter regardless of the organ considered, suggesting that the turn-over of respiratory pools strictly followed photosynthetic input. However, carbon turn-over was nearly three times greater than N turn-over in total organic matter, suggesting that new organic material synthesised was less N-rich than pre-existing organic material (due to progressive nitrogen elemental dilution) or that N remobilisation occurred to sustain growth. This pattern was not consistent with the total commitment into free amino acids where the input of new C and N appeared to be stoichiometric. The labelling pattern in Asn was complex, with contrasted C and N commitments in different organs, suggesting that neosynthesis and redistribution of new Asn molecules required metabolic remobilisation. We conclude that the production of new organic material during alfalfa growth depends on both C and N remobilisation in different organs. At the plant level, this remobilisation is complicated by allocation and metabolism in the different organs.
Additional keywords: carbon exchange, carbon isotopes, nitrogen fixation, nitrogen 15 isotope, photosynthesis, respiration.
References
Allaway D, Lodwing E, Crompton LA, Wood M, Parsons R, Wheeler TR, Poole P (2000) Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids. Molecular Microbiology 36, 508–515.| Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXjtlKitrc%3D&md5=0c4c1b5375412b7af61321b3590d7470CAS | 10792736PubMed |
Aranjuelo I, Pardo A, Biel C, Save R, Azcon-Bieto J, Nogués S (2009) Leaf carbon management in slow-growing plants exposed to elevated CO2. Global Change Biology 15, 97–109.
| Leaf carbon management in slow-growing plants exposed to elevated CO2.Crossref | GoogleScholarGoogle Scholar |
Aranjuelo I, Tcherkez G, Molero G, Françoise G, Avice JC, Nogués S (2013) Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction. Journal of Experimental Botany 64, 1–17.
| Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXjtlGrs7w%3D&md5=178e141d93357434a792e65669451435CAS |
Arrese-Igor C, Gonzalez EM, Gordon AJ, Minchin FR, Galvez L, Royuela M, Cabrerizo PM, Aparicio-Tejo PM (1999) Sucrose synthase and nodule nitrogen fixation under drought and other environmental stresses. Symbiosis 27, 189–212.
Avice JC, Ourry A, Lemaire G, Boucaud J (1996) Nitrogen and carbon flows estimated by 15N and 13C pulse–chase labelling during regrowth of alfalfa. Plant Physiology 112, 281–290.
Day DA, Poole P, Tyerman SD, Rosendahl L (2001) Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules. Cellular and Molecular Life Sciences 58, 61–71.
| Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXhslKnsr4%3D&md5=b287c79dd82dcc614d8e695152dde979CAS | 11229817PubMed |
Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology 40, 503–537.
| Carbon isotope discrimination and photosynthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXktlKmu70%3D&md5=1b7b578571b22881f7134e65a1c8f68dCAS |
Fischinger SA, Schulze J (2010) The importance of nodule CO2 fixation for the efficiency of symbiotic nitrogen fixation in pea at vegetative growth and during pod formation. Journal of Experimental Botany 61, 2281–2291.
| The importance of nodule CO2 fixation for the efficiency of symbiotic nitrogen fixation in pea at vegetative growth and during pod formation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXmslSqtLw%3D&md5=34c11f96fcdcde4971fe74a9e88f8c4dCAS | 20363863PubMed |
Godin JP, Fay LB, Hopfgartner G (2007) Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research. Mass Spectrometry Reviews 26, 751–774.
| Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtlyrt7bP&md5=e72f5341887b6cbd830575dea5909b36CAS | 17853432PubMed |
Gordon AJ, Minchin FR, James CL, Komina O (1999) Sucrose synthase in legume nodules is essential for nitrogen fixation. Plant Physiology 120, 867–878.
| Sucrose synthase in legume nodules is essential for nitrogen fixation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXks1amur8%3D&md5=65362399987a924d6972f0230071d503CAS | 10398723PubMed |
Groat RG, Vance CP (1981) Root nodule enzymes of ammonia assimilation in Alfalfa (Medicago sativa L.). Plant Physiology 67, 1198–1203.
| Root nodule enzymes of ammonia assimilation in Alfalfa (Medicago sativa L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3MXksl2itro%3D&md5=358bb087131c5bccc3cc4e5b28a5ab65CAS | 16661836PubMed |
Hardy RWF, Havelka UD (1976) Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In ‘Symbiotic nitrogen fixation’. (Ed. PS Nutman) pp. 421–439. (Cambridge University Press: Cambridge, UK)
Herrada G, Puppo A, Rigaud J (1989) Uptake of metabolites by bacteriod-containing vesicles and by free bacteroids from French bean nodules. Journal of General Microbiology 135, 3165–3177.
Heytler PG, Reddy GS, Hardy RWF (1985) In vivo energetic of symbiotic nitrogen fixation in soybeans. In ‘Nitrogen fixation and CO2 metabolism’, (Eds PL Ludden, JE Berris) pp. 283–292. (Elsevier: New York)
Kohl DH, Schubert KR, Carter MB, Hagedorn CH, Shearaer G (1988) Proline metabolism in N2-fixing nodules: energy transfer and regulation of purine synthesis. Proceedings of the National Academy of Sciences of the United States of America 85, 2036–2040.
| Proline metabolism in N2-fixing nodules: energy transfer and regulation of purine synthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXitF2jtr0%3D&md5=7572e31e9c7c2376dd016b2716c29f3bCAS | 3353366PubMed |
Lemaire G, Khaity M, Onillon B, Allirand JM, Chartier M, Gosse G (1992) Dynamics of accumulation and partitioning of N leaves, stems and roots of lucerne in a dense canopy. Annals of Botany 70, 429–435.
Lluch C, Carrero JJ, Tejera N, Ocaña A (2002) Interacción planta-microorganismo del suelo: simbiosis Fijadoras de Nitrógeno. In ‘Ecofisiología Vegetal’. (Eds JM Reigosa, N Pedrol, A Sánchez) pp. 331–360. (Paraninfo: Madrid)
Lodwig EM, Hosie AHF, Bourdes A, Findlay K, Allaway D, Karunakaran R, Downie JA, Poole P (2003) Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis. Nature 422, 722–726.
| Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXivFGhs7k%3D&md5=b1f35e3b09c42a6dff7b49a68bc38525CAS | 12700763PubMed |
Marino D, González EM, Arrese-Igor C (2006) Drought effects on carbon and nitrogen metabolism of pea nodules can be mimicked by paraquat: evidence for the occurrence of two regulation pathways under oxidative stresses. Journal of Experimental Botany 57, 665–673.
| Drought effects on carbon and nitrogen metabolism of pea nodules can be mimicked by paraquat: evidence for the occurrence of two regulation pathways under oxidative stresses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XovVCqtg%3D%3D&md5=f25faee046429bffa5c74b9716dbed22CAS | 16415332PubMed |
Matt P, Geiger M, Walch-Liu P, Engels C, Krapp A, Stitt M (2001) Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when growing on ammonium nitrate. Plant, Cell & Environment 24, 1119–1137.
| Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when growing on ammonium nitrate.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXovVKgsbY%3D&md5=929cc2c6278438e84000a54bf37457b6CAS |
Maxwell CA, Vance CP, Heichel GH, Stade S (1984) CO2 fixation in alfalfa and birdsfoot trefoil root nodules and partitioning of 14C to the plant. Crop Science 24, 257–264.
| CO2 fixation in alfalfa and birdsfoot trefoil root nodules and partitioning of 14C to the plant.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2cXitVKmt7k%3D&md5=9c563e1cbc2644209d2f3e74f60f5319CAS |
McNeill AM, Hood RC, Wood M (1994) Direct measurement of nitrogen-fixation by Trifolium-repens L and Alnus-glutinosa L. using 15N2. Journal of Experimental Botany 45, 749–755.
| Direct measurement of nitrogen-fixation by Trifolium-repens L and Alnus-glutinosa L. using 15N2.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXmsFWnt7o%3D&md5=c45a80259743d7b907a4559ebed279a9CAS |
McRae DG, Miller RW, Berndt WB, Joy K (1989) Transport of C(4)-cicarboxylates and amino acids by Rhizobium meliloti bacteroids. Molecular Plant-Microbe Interactions 2, 273–278.
| Transport of C(4)-cicarboxylates and amino acids by Rhizobium meliloti bacteroids.Crossref | GoogleScholarGoogle Scholar |
Minchin FR, Summerfield RJ, Hadley P, Roberts EH, Rawthorne S (1981) Carbon and nitrogen nutrition of nodulated roots of grain legumes. Plant, Cell & Environment 4, 5–26.
| Carbon and nitrogen nutrition of nodulated roots of grain legumes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3MXhsFGmsrw%3D&md5=99c951fc49f4372f9a5e146b83ea07abCAS |
Molero G, Aranjuelo I, Teixidor P, Araus JL, Nogués S (2011) Measurement of 12C and 15N isotope enrichment by gas chromatography-combustion-isotope ratio mass spectrometry to study amino acid fluxes. Application to a symbiotic plant - microbe association. Rapid Communications in Mass Spectrometry 25, 599–607.
| Measurement of 12C and 15N isotope enrichment by gas chromatography-combustion-isotope ratio mass spectrometry to study amino acid fluxes. Application to a symbiotic plant - microbe association.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsFCgtLg%3D&md5=b0488202ead03e5c2355cdb97b2a052aCAS | 21290446PubMed |
Nogués S, Tcherkez G, Cornic G, Ghashgaie J (2004) Respiratory carbon metabolism following illumination in intact French bean leaves using 13C/12C isotope labelling. Plant Physiology 136, 3245–3254.
| Respiratory carbon metabolism following illumination in intact French bean leaves using 13C/12C isotope labelling.Crossref | GoogleScholarGoogle Scholar | 15377781PubMed |
Owen SF, McCarthy ID, Watt PW (1999) In vivo rates of protein synthesis in Atlantic salmon (Salmo salar L.) smolts determined using a stable isotope flooding dose technique. Fish Physiology and Biochemistry 20, 87–94.
| In vivo rates of protein synthesis in Atlantic salmon (Salmo salar L.) smolts determined using a stable isotope flooding dose technique.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjtFCgs7o%3D&md5=aea65396c6924d47400957a26aa2836bCAS |
Pate JS, Atkins CA (1983) Nitrogen uptake, transport and utilization. In ‘Nitrogen fixation. Vol. 3. Legumes’. (Ed. WJ Broughton) pp. 245–298. (Oxford University Press: Oxford)
Poole P, Allaway D (2000) Carbon and nitrogen metabolism in Rhizobium. Advances in Microbial Physiology 43, 117–163.
| Carbon and nitrogen metabolism in Rhizobium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXls1ygtL0%3D&md5=615661b275007e107830080f1d8ac959CAS | 10907556PubMed |
Prell J, Poole P (2006) Metabolic changes of rhizobia in legume nodules. Trends in Microbiology 14, 161–168.
| Metabolic changes of rhizobia in legume nodules.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xjs1Gltrg%3D&md5=cb41402e01769966fee8e12e5c9e92a8CAS | 16520035PubMed |
Schmitt A, Pausch J, Kuzyakov Y (2013) C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling. Plant and Soil 368, 581–590.
| C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXps1GnsLw%3D&md5=fba992f37d31d8aaf14e3109e3c129faCAS |
Steunou AS, Jensen SI, Brecht E, Becraft ED, Bateson MM, Kilian O, Bhaya D, Ward DM, Peters JW, Grossman AR, Kühl M (2008) Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat. ISME Journal 2, 364–378.
Summerfield RJ, Minchin FR, Wien HC (1979) Screening soyabean and cowpea. An improved strategy. World Crops 31, 21–23.
Ta TC, MacDowall FDH, Faris MA (1988) Metabolism of nitrogen fixed by nodules of alfalfa (Medicago sativa L.). II. Asparagine synthesis. Biochemistry and Cell Biology 66, 1349–1354.
| Metabolism of nitrogen fixed by nodules of alfalfa (Medicago sativa L.). II. Asparagine synthesis.Crossref | GoogleScholarGoogle Scholar |
Temple SJ, Vance CP, Gantt JS (1998) Glutamate synthase and nitrogen assimilation. Trends in Plant Science 3, 51–56.
| Glutamate synthase and nitrogen assimilation.Crossref | GoogleScholarGoogle Scholar |
Udvardi MK, Day DA (1997) Metabolite transport across symbiotic membranes of legume nodules. Annual Review of Plant Physiology and Plant Molecular Biology 48, 493–523.
| Metabolite transport across symbiotic membranes of legume nodules.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXjs1emt7g%3D&md5=df49d9d9b8f502845bd984a243f7575eCAS | 15012272PubMed |
Udvardi MK, Salom CL, Day DA (1988) Transport of L-glutamate across the bacteroid membrane but not the peribacteroid membrane from soybean root nodules. Molecular Plant-Microbe Interactions 1, 250–254.
| Transport of L-glutamate across the bacteroid membrane but not the peribacteroid membrane from soybean root nodules.Crossref | GoogleScholarGoogle Scholar |
Vance CP, Heichel GH (1991) Carbon in N2 fixation: limitation or exquisite adaptation. Annual Review of Plant Physiology and Plant Molecular Biology 42, 373–390.
| Carbon in N2 fixation: limitation or exquisite adaptation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXltFSms78%3D&md5=2f9b026161292ae8759e6e400ec016e2CAS |
Voisin AS, Salon C, Jeudy C, Warembourg FR (2003) Root and nodule growth in Pisum sativum L. in relation to photosynthesis: analysis using 13C-labelling. Annals of Botany 92, 557–563.
| Root and nodule growth in Pisum sativum L. in relation to photosynthesis: analysis using 13C-labelling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXoslOmtb8%3D&md5=53066a8a5563b4a1e5b99d49de4d10ceCAS | 14507741PubMed |
Waters JK, Hughes BL, Purcell LC, Gerhardt KO, Mawhinney TP, Emerich DW (1998) Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids. Proceedings of the National Academy of Sciences of the United States of America 95, 12038–12042.
| Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXmsV2ht7c%3D&md5=f675050ef2ef7ad95685939aa0583796CAS | 9751786PubMed |
White J, Prell J, James EK, Poole P (2007) Nutrient sharing between symbionts. Plant Physiology 144, 604–614.
| Nutrient sharing between symbionts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmvValsb4%3D&md5=f56190a9aa69d4899d2a5f59ccef6d64CAS | 17556524PubMed |
