Abstract
A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.
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References
Ahmed Z (1993) Potential for expanding cotton production in Pakistan. Central Cotton Research Institute, Multan, p 114
Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8:115–118
An G (1987) Binary TI vectors for plant transformation and promoter analysis. Methods Enzymol 153:292–293
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye banding. Anal Biochem 72:248–254
Bucher M, Brunner S, Zimmermann P, Zardi GI, Amrhein N, Willmitzer L, Riesmeier JW (2002) The expression of an extensin-like protein correlates with cellular tip growth in tomato. Plant Physiol 128:911–923
George TS, Richardson AE (2008) Potential and limitations to improving crops for enhanced phosphorus utilization. In: White PJ, Hammond JP (eds) Ecophysiology of plant–phosphorus interactions. Springer, Dordrecht, pp 247–270
George TS, Richardson AE, Hadobas PA, Simpson RJ (2004) Characterisation of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth and P nutrition in laboratory media and soil. Plant Cell Environ 27:1351–1361
George TS, Richardson AE, Simpson RJ (2005a) Behaviour of plant-derived extracellular phytase upon addition to soil. Soil Biol Biochem 37:977–988
George TS, Simpson RJ, Hadobas PA, Richardson AE (2005b) Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition in plants grown in amended soil. Plant Biotechnol J 3:129–140
Gould JH, Magallanes-Cedeno M (1998) Adaptation of cotton shoot apex culture to Agrobacterium-mediated transformation. Plant Mol Biol Rep 16:1–10
Graf E, Empson KL, Eaton JW (1987) Phytic acid. A natural antioxidant. J Biol Chem 262:11647–11650
Keller G, Spatola L, Mccabe D, Martinell B, Swain W, John M (1997) Transgenic cotton resistant to herbicide bialaphos. Transgenic Res 6:385–392
Kumar M, Shukla AK, Singh H, Tuli R (2009) Development of insect resistant transgenic cotton lines expressing cry1EC gene from an insect bite and wound inducible promoter. J Biotechnol 140:143–148
Li GL (2001) The secretion phytase of transgenic soybean [D]. PhD thesis of Nankai University
Li J, Hegeman CE, Hanlon RW, Lacy GH, Denbow DM, Grabau EA (1997) Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol 114:1103–1111
Li GL, Zhu JH, Sun J, Wu ZW, Chen J, Yan J, Wang LF, Chen G, Jiang HZ, Li MG (2003) Cloning of the phytase gene phyA from Aspergillus ficuum 3.4322 and its expression in yeast. Fungal Divers 13:85–93
Marin E, Divol F, Bechtold N, Vavasseur A, Nussaume L, Forestier C (2006) Molecular characterization of three Arabidopsis soluble ABC proteins which expression is induced by sugars. Plant Sci 171:84–90
Marschner H (1995) Mineral nutrition of higher plants. Academic Press/ Harcourt Brace and Company Publishers, London, pp 483–507
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
Mullins GL (1993) Cotton root growth as affected by P fertilizer placement. Fertil Res 34:23–26
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol 15:473–497
Murphy J, Riley J (1962) A modified single solution for the determination of phosphate natural waters. Anal Chim Acta 27:31–36
Nitz I, Berkefeld H, Puzio PS, Grundler FMW (2001) Pyk10, a seedling and root specific gene and promoter from Arabidopsis thaliana. Plant Sci 161:337–346
Rana JI, Attn MR, Waheed T, Iftikhar A (2001) Phenotypic differences among cotton genotypes for phosphorus use efficiency and stress factor. Int J Agric Biol 3:186–187
Richardson AE, Hadobas PA, Hayes JE (2000) Phosphomonoesterase and phytase activities of wheat (Triticum aestivum L.) roots and utilisation of organic phosphorus substrates by seedlings grown in sterile culture. Plant Cell Environ 23:397–405
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
Richardson AE, George TS, Jakobsen I, Simpson RJ (2007) Plant utilization of inositol phosphates. In: Turner BL, Richardson AE, Mullaney EJ (eds) Inositol phosphates: linking agriculture and the environment. CAB International, Wallingford, pp 242–260
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification, and potential biotechnological applications. Crit Rev Biotechnol 23:29–60
Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, LAP van (1999) Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases):molecular size, glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 65:359–366
Xiao K, Harrison MJ, Wang ZY (2005) Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis. Planta 222:27–36
Zimmermann P, Zardi GI, 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
Acknowledgments
This research was supported by the National 973 Project (No. 2010CB126000), Project (No. 2009ZX08005-021B) from the Ministry of Agriculture of China for transgenic research and the Natural Science Foundation of Hebei Province (No. C2006001034).
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Liu, J.F., Zhao, C.Y., Ma, J. et al. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) with a fungal phytase gene improves phosphorus acquisition. Euphytica 181, 31–40 (2011). https://doi.org/10.1007/s10681-011-0370-9
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DOI: https://doi.org/10.1007/s10681-011-0370-9