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Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis

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Abstract

A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume Medicago truncatula. The phytase cDNA (MtPHY1) has an open reading frame of 1,632 bp predicted to encode 543 amino acids including an N-terminal signal peptide of 27 amino acids. The MtPHY1 gene is 5,151 bp in length, containing 7 exons and 6 introns. MtPHY1 transcripts were detected in leaves and roots and levels elevated in roots during growth in low phosphate conditions. Transgenic Arabidopsis lines expressing MtPHY1 under the control of the root-specific MtPT1 promoter or the constitutive CaMV35S promoter were created. Phytase activities in root apoplast of the transgenic Arabidopsis were 12.3- to 16.2-fold higher than those of the control plants. The expressed phytase was secreted into the rhizosphere as demonstrated by enzyme activity staining and HPLC analysis of phytate degradation by root exudates. Transgenic expression of the MtPHY1 led to significant improvement in organic phosphorus utilization and plant growth. When phytate was supplied as the sole source of phosphorus, dry weight of the transgenic Arabidopsis lines were 3.1- to 4.0-fold higher than the control plants and total phosphorus contents were 4.1- to 5.5-fold higher than the control. Transgenic expression of phytase genes of plant origin has great potential for improving plant phosphorus acquisition and for phytoremediation.

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Abbreviations

EST :

Expression sequence tag

GFP :

Green fluorescent protein

GMO :

Genetically modified organism

GUS :

Glucuronidase

MtPHY1 :

Medicago truncatula phytase 1

MtPT1 :

Medicago truncatula phosphate transporter 1

ORF :

Open reading frame

P :

Phosphorus

PAP :

Purple acid phosphatase

Pi :

Inorganic phosphate

PPT :

Phosphinothricin

RACE :

Rapid amplification of cDNA ends

References

  • Abelson PH (1999) A potential phosphate crisis. Science 283:2015

    Article  PubMed  CAS  Google Scholar 

  • Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. In: Neufeld E, Ginsburg V (eds) Methods in enzymology. Academic Press, New York, pp 115–118

    Google Scholar 

  • Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  PubMed  Google Scholar 

  • Bieleski RL (1973) Phosphate pools, phosphate transport, and phosphate availability. Annu Rev Plant Physiol 24:225–252

    Article  CAS  Google Scholar 

  • Brinch-Pedersen H, Olesen A, Rasmussen SK, Holm PB (2000) Generation of transgenic wheat (Triticum aestivum L) for constitutive accumulation of an Aspergillus phytase. Mol Breed 6:195–206

    Article  CAS  Google Scholar 

  • Brinch-Pedersen H, Sorensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125

    Article  PubMed  CAS  Google Scholar 

  • Brinch-Pedersen H, Hatzack F, Sorensen L, Holm P (2003) Concerted action of endogenous and heterologous phytase on phytic acid degradation in seed of transgenic wheat (Triticum aestivum L). Transgenic Res 12:649–659

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16:664–670

    PubMed  CAS  Google Scholar 

  • Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Dalal RC (1977) Soil organic phosphorus. Adv Agron 29:83–117

    Article  CAS  Google Scholar 

  • Denbow DM, Grabau EA, Lacy GH, Kornegay ET, Russell DR, Umbeck PF (1998) Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers. Poultry Sci 77:878–881

    CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016

    Article  PubMed  CAS  Google Scholar 

  • Gaston LA, Drapcho CM, Tapadar S, Kovar JL (2003) Phosphorus runoff relationships for Louisiana Coastal Plain soils amended with poultry litter. J Environ Qual 32:1422–1429

    Article  PubMed  CAS  Google Scholar 

  • Hayes JE, Richardson AE, Simpson RJ (1999) Phytase and acid phosphatase activities in extracts from roots of temperate pasture grass and legume seedlings. Aust J Plant Physiol 26:801–809

    Article  CAS  Google Scholar 

  • Hayes JE, Simpson RJ, Richardson AE (2000) The growth and phosphorus utilisation of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate. Plant Soil 220:165–174

    Article  CAS  Google Scholar 

  • Hegeman CE, Grabau EA (2001) A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol 126:1598–1608

    Article  PubMed  CAS  Google Scholar 

  • Holford ICR (1997) Soil phosphorus: its measurement, and its uptake by plants. Aust J Soil Res 35:227–239

    Article  CAS  Google Scholar 

  • Hong C-Y, Cheng K-J, Tseng T-H, Wang C-S, Liu L-F, Yu S-M (2004) Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 13:29–39

    Article  PubMed  CAS  Google Scholar 

  • Hübel F, Beck E (1996) Maize root phytase. Purification, characterization, and localization of enzyme activity and its putative substrate. Plant Physiol 112:1429–1436

    PubMed  Google Scholar 

  • Iyamuremye F, Dick RP (1996) Organic amendments and phosphorus sorption by soils. Adv Agron 56:139–185

    Article  CAS  Google Scholar 

  • Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291

    Article  PubMed  CAS  Google Scholar 

  • König A (2003) A framework for designing transgenic crops—science, safety and citizen’s concerns. Nat Biotechnol 21:1274–1279

    Article  PubMed  Google Scholar 

  • Larsen S (1967) Soil phosphorus. Adv Agron 19:151–210

    Article  CAS  Google Scholar 

  • Li J, Hegeman CE, Hanlon RW, Lacy GH, Denbow DM, Grabau EA (1997a) Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol 114:1103–1111

    Article  PubMed  CAS  Google Scholar 

  • Li M, Osaki M, Rao IM, Tadano T (1997b) Secretion of phytase from the roots of several plant species under phosphorus-deficient conditions. Plant Soil 195:161–169

    Article  Google Scholar 

  • Liu H, Trieu AT, Blaylock LA, Harrison MJ (1998) Cloning and characterization of two phosphate transporters from Medicago truncatula roots: Regulation in response to phosphate and to colonization by arbuscular mycorrhizal (AM) fungi. Mol Plant Microbe Interact 11:14–22

    Article  PubMed  CAS  Google Scholar 

  • Lucca P, Hurrell R, Potrykus I (2001a) Approaches to improving the bioavailability and level of iron in rice seeds. J Sci Food Agric 81:828–834

    Article  CAS  Google Scholar 

  • Lucca P, Hurrell R, Potrykus I (2001b) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392–397

    Article  CAS  Google Scholar 

  • Matuszeski W (2000) Phosphorus and the Chesapeake Bay—opening remarks. In: Sharpley AN (ed) Agriculture and phosphorus management: the Chesapeake Bay. Lewis Publishers, Boca Raton, pp 3–5

    Google Scholar 

  • Maugenest S, Martinez I, Lescure AM (1997) Cloning and characterization of a cDNA encoding a maize seedling phytase. Biochem J 322:511–517

    PubMed  CAS  Google Scholar 

  • Maugenest S, Martinez I, Godin B, Perez P, Lescure AM (1999) Structure of two maize phytase genes and their spatio-temporal expression during seedling development. Plant Mol Biol 39:503–514

    Article  PubMed  CAS  Google Scholar 

  • Miller SS, Liu J, Allan DL, Menzhuber CJ, Fedorova M, Vance CP (2001) Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin. Plant Physiol 127:594–606

    Article  PubMed  CAS  Google Scholar 

  • Mudge SR, Smith FW, Richardson AE (2003) Root-specific and phosphate-regulated expression of phytase under the control of a phosphate transporter promoter enables Arabidopsis to grow on phytate as a sole P source. Plant Sci 165:871–878

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36

    Article  CAS  Google Scholar 

  • Nielsen KM (2003) Transgenic organisms—time for conceptual diversification? Nat Biotechnol 21:227–228

    Article  PubMed  CAS  Google Scholar 

  • Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Article  PubMed  CAS  Google Scholar 

  • Pant HK, Adjei MB, Scholberg JMS, Chambliss CG, Rechcigl JE (2004) Forage production and phosphorus phytoremediation in manure-impacted soils. Agron J 96:1780–1786

    Article  Google Scholar 

  • Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J (2002) Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol Breed 10:31–44

    Article  CAS  Google Scholar 

  • Pote DH, Kingery WL, Aiken GE, Han FX, Moore PAJ, Buddington K (2003) Water-quality effects of incorporating poultry litter into perennial grassland soils. J Environ Qual 32:2392–2398

    Article  PubMed  CAS  Google Scholar 

  • Richardson AE, Hadobas PA, Hayes JE (2000) Acid phosphomonoesterase and phytase activities of wheat (Triticum aestivum L) roots and utilization of organic phosphorus substrates by seedlings grown in sterile culture. Plant Cell Environ 23:397–405

    Article  CAS  Google Scholar 

  • Richardson AE, Hadobas PA, Hayes JE (2001) Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant J 25:641–649

    Article  PubMed  CAS  Google Scholar 

  • Roe BA, Kupfer DM (2004) Sequencing gene rich regions of Medicago truncatula, a model legume. In: Hopkins A, Wang Z-Y, Mian R, Sledge M, Barker RE (eds) Molecular Breeding of Forage and Turf. Kluwer, Dordrecht, pp 333–344

    Chapter  Google Scholar 

  • Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang L, Perry R, Swords K (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135:421–431

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Ullah AHJ, Dischinger HCJ (1993) Aspergillus ficuum phytase: complete primary structure elucidation by chemical sequencing. Biochem Biophys Res Comm 192:747–753

    Article  PubMed  CAS  Google Scholar 

  • Vance CP, Uhde SC, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423–447

    Article  CAS  Google Scholar 

  • Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification, and potential biotechnological applications. Crit Rev Biotechnol 23:29–60

    Article  PubMed  CAS  Google Scholar 

  • Wodzinski RJ, Ullah AHJ (1996) Phytase. Adv Appl Microbiol 42:263–302

    Article  PubMed  CAS  Google Scholar 

  • Yip W, Wang L, Cheng C, Wu W, Lung S, Lim B-L (2003) The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco. Biochem Biophys Res Comm 310:1148–1154

    Article  PubMed  CAS  Google Scholar 

  • Yu Q, Tang C, Chen Z, Kuo J (1999) Extraction of apoplastic sap from plant roots by centrifugation. New Phytol 143:299–304

    Article  Google Scholar 

  • Zimmermann P, Zardi G, Lehmann M, Zeder C, Amrhein N, Frossard E, Bucher M (2003) Engineering the root-soil interface via targeted expression of a synthetic phytase gene in trichoblasts. Plant Biotechnol J 1:353–360

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Xuefeng Ma for critically reading the manuscript and Caroline Lara for editing it. The work was supported by the Samuel Roberts Noble Foundation.

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  1. Kai Xiao

    Present address: College of Agronomy, Hebei Agricultural University, Baoding, 071001, China

Authors and Affiliations

  1. Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    Kai Xiao & Zeng-Yu Wang

  2. Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY, 14853-1801, USA

    Maria J. Harrison

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  1. Kai Xiao
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  2. Maria J. Harrison
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Correspondence to Zeng-Yu Wang.

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Xiao, K., Harrison, M.J. & Wang, ZY. Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis . Planta 222, 27–36 (2005). https://doi.org/10.1007/s00425-005-1511-y

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