Plant and Soil

, Volume 353, Issue 1–2, pp 221–230 | Cite as

Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene

  • F. Nascimento
  • C. Brígido
  • L. Alho
  • B. R. Glick
  • S. Oliveira
Regular Article



The main goal of the study reported herein was to assess the nodulation performance of a Mesorhizobium strain transformed with an exogenous ACC deaminase gene (acdS), and its subsequent ability to increase chickpea plant growth under normal and waterlogged conditions.


The Mesorhizobium ciceri strain LMS-1 was transformed with the acdS gene of Pseudomonas putida UW4 by triparental conjugation using plasmid pRKACC. A plant growth assay was conducted to verify the plant growth promotion ability of the LMS-1 (pRKACC) transformed strain under normal and waterlogging conditions. Bacterial ACC deaminase and nitrogenase activity was measured.


By expressing the exogenous acdS gene, the transformed strain LMS-1 showed a 127% increased ability to nodulate chickpea and a 125% promotion of the growth of chickpea compared to the wild-type strain, under normal conditions. Plants inoculated with the LMS-1 wild-type strain showed a higher nodule number under waterlogging stress than under control conditions, suggesting that waterlogging increases nodulation in chickpea. No significant relationship was found between ACC deaminase and nitrogenase activity.


The results obtained in this study show that the use of rhizobial strains with improved ACC deaminase activity might be very important for developing microbial inocula for agricultural purposes.


Rhizobia Chickpea ACC deaminase Waterlogging Nodulation 



The research leading to these results has received funding from Fundação para a Ciência e a Tecnologia (FCT) and co-financed by FEDER (PTDC/BIO/80932/2006) and from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 247669. C. Brígido acknowledges a FCT fellowship (SFRH/BD/30680/2006). The authors thank G. Mariano for technical assistance. Chickpea seeds were generously provided by the Instituto Nacional de Recursos Biológicos, Elvas, Portugal.


  1. Abeles F, Morgan P, Saltveit M Jr (1992) Ethylene in plant biology, 2nd edn. Academic, New YorkGoogle Scholar
  2. Alexandre A, Brígido C, Laranjo M, Rodrigues S, Oliveira S (2009) Survey of chickpea rhizobia diversity in Portugal reveals the predominance of species distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum. Microb Ecol 58(4):930–941PubMedCrossRefGoogle Scholar
  3. Banga M, Bogemann GM, Blom CWPM, Voesenek LACJ (1997) Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence. Physiol Plantarum 99(3):415–422CrossRefGoogle Scholar
  4. Belimov AA, Safronova VI, Sergeyeva TA, Egorova TN, Matveyeva VA, Tsyganov VE, Borisov AY, Tikhonovich IA, Kluge C, Preisfeld A, Dietz KJ, Stepanok VV (2001) Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase. Can J Microbiol 47(7):642–652PubMedCrossRefGoogle Scholar
  5. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198PubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Bradford KJ, Dilley DR (1978) Effects of root anaerobiosis on ethylene production, epinasty, and growth of tomato plants. Plant Physiol 61(4):506–509PubMedCrossRefGoogle Scholar
  8. Bradford KJ, Hsiao TC, Yang SF (1982) Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress. Plant Physiol 70(5):1503–1507PubMedCrossRefGoogle Scholar
  9. Broughton WJ, Dilworth MJ (1971) Control of leghaemoglobin synthesis in snake beans. Biochem J 125(4):1075–1080PubMedGoogle Scholar
  10. Caba JM, Recalde L, Ligero F (1998) Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa. Plant Cell Environ 21(1):87–93CrossRefGoogle Scholar
  11. Conforte VP, Echeverria M, Sanchez C, Ugalde RA, Menendez AB, Lepek VC (2010) Engineered ACC deaminase-expressing free-living cells of Mesorhizobium loti show increased nodulation efficiency and competitiveness on Lotus spp. J Gen Appl Microbiol 56(4):331–338PubMedCrossRefGoogle Scholar
  12. Cowie AL, Jessop RS, MacLeod DA (1996a) Effects of waterlogging on chickpeas.1. Influence of timing of waterlogging. Plant Soil 183(1):97–103CrossRefGoogle Scholar
  13. Cowie AL, Jessop RS, MacLeod DA (1996b) Effects of waterlogging on chickpeas.2. Possible causes of decreased tolerance of waterlogging at flowering. Plant Soil 183(1):105–115CrossRefGoogle Scholar
  14. Delgado MJ, Bedmar EJ, Downie JA (1998) Genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation. Adv Microb Physiol 40:191–231PubMedCrossRefGoogle Scholar
  15. Duan J, Muller KM, Charles TC, Vesely S, Glick BR (2008) 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes in rhizobia from southern Saskatchewan. Microb Ecol 57(3):423–436PubMedCrossRefGoogle Scholar
  16. Duarte I, De Sousa M, Pereira M, Carita T (1992) Duas novas cultivares de grão-de-bico para sementeira antecipada de Outono: ELMO e ELVAR. Pastagens e Forragens 13:125–134Google Scholar
  17. Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM (2010) Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52(1):61–76PubMedCrossRefGoogle Scholar
  18. Finan TM, Kunkel B, Devos GF, Signer ER (1986) 2nd symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167(1):66–72PubMedGoogle Scholar
  19. Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen−fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 68(2):280–300PubMedCrossRefGoogle Scholar
  20. Gallacher A, Sprent J (1978) The effect of different water regimes on growth and nodule development of greenhouse−grown Vicia faba. J Exp Botany 29(2):413–423CrossRefGoogle Scholar
  21. Glick BR (2003) Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnol Adv 21(5):383–393PubMedCrossRefGoogle Scholar
  22. Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theor Biol 190(1):63–68PubMedCrossRefGoogle Scholar
  23. Grichko VP, Glick BR (2001) Amelioration of flooding stress by ACC deaminase-containing plant growth-promoting bacteria. Plant Physiol Bioch 39(1):11–17CrossRefGoogle Scholar
  24. Guinel FC, Geil RD (2002) A model for the development of the rhizobial and arbuscular mycorrhizal symbioses in legumes and its use to understand the roles of ethylene in the establishment of these two symbioses. Can J Bot 80(7):695–720CrossRefGoogle Scholar
  25. Hong TD, Minchin FR, Summerfield RJ (1977) Recovery of nodulated cowpea plants (Vigna unguiculata (L.) Walp.) from waterlogging during vegetative growth. Plant Soil 48(3):661–672CrossRefGoogle Scholar
  26. Jackson MB (1985) Ethylene and responses of plants to soil waterlogging and submergence. Annu Rev Plant Phys 36:145–174CrossRefGoogle Scholar
  27. Jackson MB, Campbell DJ (1976) Waterlogging and petiole epinasty in tomato - role of ethylene and low oxygen. New Phytologist 76(1):21–29CrossRefGoogle Scholar
  28. Kozlowski TT (1984) Extent, causes, and impacts of flooding. In: Kozlowski TT (ed) Flooding and plant growth. Academic, New York, pp 1–5Google Scholar
  29. Ma WB, Guinel FC, Glick BR (2003a) Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants. Appl Environ Micro 69(8):4396–4402CrossRefGoogle Scholar
  30. Ma WB, Sebestianova SB, Sebestian J, Burd GI, Guinel FC, Glick BR (2003b) Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp. Anton Leeuw Int J G 83(3):285–291CrossRefGoogle Scholar
  31. Ma WB, Charles TC, Glick BR (2004) Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa. Appl Environ Micro 70(10):5891–5897CrossRefGoogle Scholar
  32. Mayak S, Tirosh T, Glick BR (2004) Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol Biochem 42(6):565–572PubMedCrossRefGoogle Scholar
  33. Mesa S, Alche JD, Bedmar EJ, Delgado MJ (2004) Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules. Physiol Plantarum 120(2):205–211CrossRefGoogle Scholar
  34. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, p 431Google Scholar
  35. Minchin FR, Pate JS (1975) Effects of water, aeration, and salt regime on nitrogen-fixation in a nodulated legume—definition of an optimum root environment. J Exp Bot 26(90):60–69CrossRefGoogle Scholar
  36. Nukui N, Minamisawa K, Ayabe S, Aoki T (2006) Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099. Appl Environ Micro 72(7):4964–4969CrossRefGoogle Scholar
  37. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plantarum 118(1):10–15CrossRefGoogle Scholar
  38. Robertsen BK, Aman P, Darvill AG, Mcneil M, Albersheim P (1981) Host-Symbiont Interactions.5. The structure of acidic extracellular polysaccharides secreted by rhizobium leguminosarum and Rhizobium trifolii. Plant Physiol 67(3):389–400PubMedCrossRefGoogle Scholar
  39. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  40. Sanchez C, Tortosa G, Granados A, Delgado A, Bedmar EJ, Delgado MJ (2011) Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding. Soil Biol Biochem 43(1):212–217CrossRefGoogle Scholar
  41. Saxena M, Singh K (1987) The chickpea. CAB International, WallingfordGoogle Scholar
  42. Schmidt JS, Harper JE, Hoffman TK, Bent AF (1999) Regulation of soybean nodulation independent of ethylene signaling. Plant Physiol 119(3):951–959PubMedCrossRefGoogle Scholar
  43. Schwinghamer MW (1994) Grower guide to identification of chickpea diseases in northern NSW. NSW Agriculture/Grains Research and Development Corporation, NSWGoogle Scholar
  44. Shah S, Li JP, Moffatt BA, Glick BR (1998) Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria. Can J Microbiol 44(9):833–843PubMedCrossRefGoogle Scholar
  45. Siddique KHM, Brinsmead RB, Knight R, Knights EJ, Paull JG, Rose IA (2000) Adaptation of chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) to Australia. In: Knight R (ed) Linking research and marketing opportunities for pulses in the 21st century, vol. 34. Current plant science and biotechnology in agriculture. Springer, Dordrecht, pp 289–303CrossRefGoogle Scholar
  46. Somasegaran P, Hoben H (1994) Handbook for rhizobia. Springer, New YorkCrossRefGoogle Scholar
  47. Sprent JI (1972) Effects of water stress on nitrogen-fixing root nodules.4. Effects on whole plants of vicia faba and glycine max. New Phytol 71(4):603–611CrossRefGoogle Scholar
  48. Uchiumi T, Ohwada T, Itakura M, Mitsui H, Nukui N, Dawadi P, Kaneko T, Tabata S, Yokoyama T, Tejima K, Saeki K, Omori H, Hayashi M, Maekawa T, Sriprang R, Murooka Y, Tajima S, Simomura K, Nomura M, Suzuki A, Shimoda Y, Sioya K, Abe M, Minamisawa K (2004) Expression islands clustered on the symbiosis island of the Mesorhizobium loti genome. J Bacteriol 186(8):2439–2448PubMedCrossRefGoogle Scholar
  49. Vartapetian BB, Jackson MB (1997) Plant adaptations to anaerobic stress. Ann Bot-London 79(suppl 1):3–20Google Scholar
  50. Wang TW, Arteca RN (1992) Effects of low O2 root stress on ethylene biosynthesis in tomato plants (Lycopersicon esculentum Mill cv Heinz 1350). Plant Physiol 98(1):97–100PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • F. Nascimento
    • 1
  • C. Brígido
    • 1
  • L. Alho
    • 1
  • B. R. Glick
    • 2
  • S. Oliveira
    • 1
    • 3
  1. 1.Laboratório de Microbiologia do Solo, I.C.A.A.M., Instituto de Ciências Agrárias e Ambientais MediterrânicasUniversidade de ÉvoraÉvoraPortugal
  2. 2.Department of BiologyUniversity of WaterlooWaterlooCanada
  3. 3.Departamento de BiologiaUniversidade de ÉvoraÉvoraPortugal

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