Skip to main content

Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa

Plant Cell Reports volume 27, pages 893–901 (2008)Cite this article

Abstract

Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants were assayed for exclusion of Al from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Overall, only the soil assays yielded consistent results. Based on the soil assays, two transgenic events were identified that were more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  • An YQ, McDowell JM, Huang SR, McKinney EC, Chambliss S, Meagher RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10:107–121. doi:10.1046/j.1365-313X.1996.10010107.x

    PubMed  Article  CAS  Google Scholar 

  • Anoop VM, Basu U, McCammon MT, McAlister-Henn L, Taylor GJ (2003) Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase. Plant Physiol 132:2205–2217

    PubMed  Article  CAS  Google Scholar 

  • Austin S, Bingham ET, Matthews BF, Shahan MN, Will J, Burgess RR (1995) Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase. Euphytica 85:381–393

    Article  CAS  Google Scholar 

  • Bergmeyer HU, Gawehn K Grassl M (1974) In: Bergmeyer HU (eds) Methods of enzymatic analysis. vol I, 2nd edn. Academic Press, New York, pp 442–443

  • Bingham ET (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research. Crop Sci 31:1098

    Article  Google Scholar 

  • Bouton JH, Sumner ME (1983) Alfalfa, Medicago sativa L., in highly weathered, acid soils. V. Field performance of alfalfa selected for acid tolerance. Plant Soil 74:431–436

    Article  CAS  Google Scholar 

  • Bouton JH, Parrott WA (1997) Salinity and aluminum stress. In: McKersie rBD, Brown DCW (eds) Biotechnology and the improvement of forage legumes. CAB International, Wallingford, New York, pp 203–226

    Google Scholar 

  • Bouton JH, Sumner ME, Hammel JE, Shahandeh H (1986) Yield of an alfalfa germplasm selected for acid soil tolerance when grown in soil with modified subsoils. Crop Sci 26:334–336

    CAS  Article  Google Scholar 

  • Buol SW, Eswaran H (1993) Assessment and conquest of poor soils. In: Maranville JW (ed) Adaptation of plants to soil stresses. Intsormil Publication No. 94–2, University of Nebraska, Lincoln, pp 17–27

    Google Scholar 

  • Covert SF, Kapoor P, Lee MH, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105(part 3):259–264

    Article  CAS  Google Scholar 

  • Dall’Agnol M, Bouton JH, Parrott WA (1996) Screening methods to develop alfalfa populations tolerant of acid, aluminum toxic soils. Crop Sci 36:64–70

    CAS  Article  Google Scholar 

  • de la Fuente JM, Ramirez-Rodriguez V, Cabrera-Ponce JL, Herrera-Estrella L (1997) Aluminum tolerance in transgenic plants by alteration of citrate synthesis. Science 276:1566–1568

    Article  Google Scholar 

  • de la Fuente JM, Herrera-Estrella L (1999) Advances in the understanding of aluminum toxicity and the development of aluminum-tolerant transgenic plants. Adv Agron 66:103–120

    Article  Google Scholar 

  • Delhaize E, Ryan PR, Randall PJ (1993) Aluminum tolerance in wheat (Triticum aestivum L).2. Aluminum-stimulated excretion of malic-acid from root apices. Plant Physiol 103:695–702

    PubMed  CAS  Google Scholar 

  • Delhaize E, Hebb DM, Ryan PR (2001) Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant Physiol 125:2059–2067

    PubMed  Article  CAS  Google Scholar 

  • Delhaize E, Ryan PR, Hebb DM, Yamamoto Y, Sasaki T, Matsumoto H (2004) Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc Natl Acad Sci USA 101:15249–15254. doi:10.1073/pnas/0406258101

    PubMed  Article  CAS  Google Scholar 

  • Devine TE, Bouton JH, Mabrahtu T (1990) Legume genetics and breeding for stress tolerance and nutrient efficiency. In: Baligar VC, Duncan RR (eds) Crops as enhancers of nutrient use. Academic Press, San Diego, pp 211–252

    Google Scholar 

  • Donald LJ, Molgat GF, Duckworth HW (1989) Cloning, sequencing and expression of the gene for NADH-sensitive citrate synthase of Pseudomona aeruginosa. J Bacteriol 171:5542–5550

    PubMed  CAS  Google Scholar 

  • Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol Biol 24:119–127

    PubMed  Article  CAS  Google Scholar 

  • Hartel PG, Bouton JH (1989) Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid, aluminum-rich soils. Plant Soil 116:283–285

    Article  CAS  Google Scholar 

  • Hoekenga OA, Maron LG, Pineros MA, Cancado GMA, Shaff J, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Yamamoto Y, Koyama H, Kochian LV (2006) AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci USA 103:9738–9743

    PubMed  Article  CAS  Google Scholar 

  • Kinraide TB (1991) Identity of the rhizotoxic aluminum species. Plant Soil 134:167–178

    CAS  Google Scholar 

  • Kochian LV (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annu Rev Plant Physiol Plant Mol Biol 46:237–260

    Article  CAS  Google Scholar 

  • Koyama H, Takita E, Kawamura A, Hara T, Shibata D (1999) Overexpression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant Cell Physiol 40:482–488

    PubMed  CAS  Google Scholar 

  • Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D (2000) Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus limited soil. Plant Cell Physiol 41:1030–1037

    PubMed  Article  CAS  Google Scholar 

  • Lopez-Bucio J, Nieto-Jacobo MF, Ramirez-Rodriguez V, Herrera-Estrella L (2000) Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci 160:1–13

    PubMed  Article  CAS  Google Scholar 

  • Ma JF, Ryan PR, Delhaize E (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci 6:273–278

    PubMed  Article  CAS  Google Scholar 

  • Matsumoto H (2000) Cell biology of Al tolerance and toxicity in higher plants. Int Rev Cytol 200:1–46

    PubMed  CAS  Article  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326

    PubMed  Article  CAS  Google Scholar 

  • Parrott WA, Bouton JH (1990) Aluminum tolerance in alfalfa as expressed in tissue culture. Crop Sci 30:387–389

    Article  Google Scholar 

  • Pineros MA, Magalhaes JV, Carvalho Alves VM, Kochian LV (2002) The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize. Plant Physiol 129:1194–1206

    PubMed  Article  CAS  Google Scholar 

  • Pineros MA, Shaff JE, Manslank HS, Alves VMC, Kochian LV (2005) Aluminum resistance in maize cannot be solely explained by root organic acid exudation. A comparative physiological study. Plant Physiol 137:231–241

    PubMed  Article  CAS  Google Scholar 

  • Polle E, Konzak CF, Kittrick JA (1978) Visual detection of aluminum tolerance levels in wheat by hematoxylin staining of seedling roots. Crop Sci 18:823–827

    CAS  Article  Google Scholar 

  • Rosellini D, Capomaccio S, Ferradini N, Savo Sardaro ML, Nicolia A, Veronesi F (2007) Non-antibiotic, efficient selection for alfalfa genetic engineering. Plant Cell Rep 26(7):1035-1044

    PubMed  Article  CAS  Google Scholar 

  • Ruijter G, Panneman H, Xu D-B, Visser J (2000) Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production. FEMS Lett 184:35–40

    CAS  Google Scholar 

  • Ryan PR, Delhaize E, Randall PJ (1995) Characterization of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196:103–110

    Article  CAS  Google Scholar 

  • Samac DA, Tesfaye M (2003) Plant improvement for tolerance to aluminum in acid soils––a review. Plant Cell Tissue Organ Cult 75:189–207

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  • Siedow JN, Day DA (2000) Respiration and photorespiration. In: Buchanan BB, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, pp 676–728

    Google Scholar 

  • Sledge MK, Bouton JH, Dall’Agnol M, Parrott WA, Kochert G (2002) Identification and confirmation of aluminum tolerance QTL in diploid Medicago sativa subsp coerulea. Crop Sci 42:1121–1128

    CAS  Article  Google Scholar 

  • Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964. doi:10.1038/35023079

    PubMed  Article  CAS  Google Scholar 

  • Tesfaye M, Temple SJ, Allan DL, Vance CP, Samac DA (2001) Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiol 127:1836–1844. doi:10.1104/pp010376

    PubMed  Article  CAS  Google Scholar 

  • von Uexkull HR, Mutert E (1995) Global extent, development and economic impact of acid soils. In: Date RA, Grundon NJ, Raymet GE, Probert ME (eds) Plant–soil interaction at low pH: principles and management. Kluwer Academic Publisher, Dordrecht, pp 5–19

    Google Scholar 

  • Yamamoto YT, Taylor GT, Acedo GN, Cheng C-L, Conkling MA (1991) Characterization of cis-acting sequences regulating root-specific gene expression in tobacco. Plant Cell 3:371–382

    PubMed  Article  CAS  Google Scholar 

  • Yang ZM, Sivaguru M, Horst WJ, Matsumoto H (2000) Aluminum resistance is achieved by exudation of citric acid from roots of soybean (Glycine max). Physiol Plant 110:72–77

    Article  CAS  Google Scholar 

  • Zhao Z, Ma JF, Sato K, Takeda K (2003) Differential Al resistance and citrate secretion in barley (Hordeum vulgare L.). Planta 17:794–800. doi:10.1007/s00425-003-1043-2

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge Monica Schmidt for generous advice, Greg Martin for valuable technical help, and Ann Bunce and Donald Wood for their help with the greenhouse plant management and soil assays. This work was funded by NATO Advanced Fellowship Programme 1999, grant #. 215.32 that supported D.R.’s stay at The University of Georgia, and by State and Hatch monies allocated to the Georgia Agricultural Experiment Stations

Author information

Author notes

  1. Pierluigi Barone

    Present address: Department of Crop Sciences, University of Illinois, 1201 W Gregory, Urbana, IL, USA

  2. Daniele Rosellini

    Present address: Dipartimento di Biologia Applicata, University of Perugia, Perugia, Italy

  3. Joseph Bouton

    Present address: The Samuel Roberts Noble Foundation, Ardmore, OK, USA

Affiliations

  1. Department of Crop and Soil Sciences, University of Georgia, 30602, Athens, GA, USA

    Pierluigi Barone, Daniele Rosellini, Peter LaFayette, Joseph Bouton & Wayne Parrott

  2. Dipartimento di Biologia Applicata, University of Perugia, Perugia, Italy

    Fabio Veronesi

Authors
  1. Pierluigi Barone
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniele Rosellini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter LaFayette
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph Bouton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fabio Veronesi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wayne Parrott
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pierluigi Barone.

Additional information

Pierluigi Barone and Daniele Rosellini contributed equally to this work.

Communicated by A. Atanassov.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Barone, P., Rosellini, D., LaFayette, P. et al. Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa. Plant Cell Rep 27, 893–901 (2008). https://doi.org/10.1007/s00299-008-0517-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-008-0517-x

Keywords

  • Acid soils
  • Aluminum toxicity
  • Citrate synthase
  • Genetic engineering
  • Medicago sativa L.
  • Transformation