Molecular Biotechnology

, Volume 59, Issue 8, pp 334–342 | Cite as

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake

  • Samreen Mohsin
  • Asma Maqbool
  • Mehwish Ashraf
  • Kauser Abdulla Malik
Original Paper

Abstract

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated To events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08–1.77, 0.02–0.67 and 0.44–2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01–1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.

Keywords

Wheat Root-specific expression Phytase Aspergillus japonicus Phosphorus Phytate 

Supplementary material

12033_2017_20_MOESM1_ESM.docx (494 kb)
Supplementary material 1 (DOCX 494 kb)

References

  1. 1.
    Almeida, J. P. F., Hartwig, U. A., Frehner, M., Nosenberger, J., & Luscher, A. (2000). Evidence that P deficiency induces feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.). Journal of Experimental Botany, 51, 1289–1297.Google Scholar
  2. 2.
    George, T. S., & Richardson, A. E. (2008). Potential and limitations to improving crops for enhanced phosphorus utilization. In P. J. White & J. P. Hammond (Eds.), Ecophysiology of plant-phosphorus interactions (pp. 247–270). Berlin: Springer.CrossRefGoogle Scholar
  3. 3.
    Wang, J., Sun, J., Miao, J., Guo, J., Shi, Z., He, M., et al. (2013). A wheat phosphate starvation response regulator Ta-PHR1 is involved in phosphate signalling and increases grain yield in wheat. Annals of Botany, 111(6), 1139–1153.CrossRefGoogle Scholar
  4. 4.
    Iyamuremye, F., Dick, R. P., & Baham, J. (1996). Organic amendment and phosphorus dynamics: II. Distribution of soil phosphorus fractions. Soil Science, 161, 436–443.CrossRefGoogle Scholar
  5. 5.
    Turner, B. L., Paphazy, M. J., Haygarth, P. M., & Mckelvie, I. D. (2002). Inositol phosphates in the environment. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 357, 449–469.CrossRefGoogle Scholar
  6. 6.
    Cade-Menun, U. (2005). Using phosphorus-31 nuclear magnetic resonance spectroscopy to characterize organic phosphorus in environmental samples. In D. S. Baldwin, B. L. Turner, & E. Frossard (Eds.), Organic phosphorus in the environment (pp. 21–44). Wallingford: CABI Publishing.CrossRefGoogle Scholar
  7. 7.
    Richardson, A. E., George, T. S., Hens, M., & Simpson, R. J. (2005). Utilization of soil organic phosphorus by higher plants. In D. S. Baldwin, B. L. Turner, & E. Frossard (Eds.), Organic phosphorus in the environment (pp. 161–184). Wallingford: CABI Publishing.Google Scholar
  8. 8.
    Belgaroui, N., Berthomieu, P., Rouached, H., & Hanin, M. (2016). The secretion of the bacterial phytase PHY-US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co-growth. Plant Biotechnology Journal, 14(9), 1914–1924.CrossRefGoogle Scholar
  9. 9.
    Bowman, B. T., Thomas, E. L., & Elrick, D. E. (1967). The movement of phytic acid in soil cores. Soil Science Society of America Journal Proceedings, 31(4), 477–481.CrossRefGoogle Scholar
  10. 10.
    McKercher, R. B., & Anderson, G. (1989). Organic phosphate sorption by neutral and basic soils. Communications in Soil Science and Plant Analysis, 20, 723–732.CrossRefGoogle Scholar
  11. 11.
    Vance, C. P., Uhde-stone, C., & Allan, D. L. (2003). Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytologist, 157, 423–447.CrossRefGoogle Scholar
  12. 12.
    Gerke, J. (2015). Phytate (inositol hexaisphosphate) in soil and phosphate acquisition from inositol phosphates by higher plants. A review. Plants., 4, 253–266.CrossRefGoogle Scholar
  13. 13.
    Childers, D. L., Corman, J., Edwards, M., & Elser, J. J. (2011). Sustainability challenges of phosphorus and food: Solutions from closing the human phosphorus cycle. BioScience, 61(2), 117–124.CrossRefGoogle Scholar
  14. 14.
    Holford, I. C. R. (1997). Soil phosphorus: Its measurement and its uptake by plants. Australian Journal of Soil Research, 35, 227–239.CrossRefGoogle Scholar
  15. 15.
    Tian, J., Wang, X., Tong, Y., Chen, X., & Liao, H. (2012). Bioengineering and management for efficient phosphorus utilization in crops and pastures. Current Opinion in Biotechnology, 23, 1–6.CrossRefGoogle Scholar
  16. 16.
    Gilroy, S., & Jones, D. L. (2000). Through form to function: Root hair development and nutrient uptake. Trends in Plant Science, 5, 56–60.CrossRefGoogle Scholar
  17. 17.
    Richardson, A. E., Hadobas, P. A., & Hayes, J. E. (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 and Environment, 23, 397–405.CrossRefGoogle Scholar
  18. 18.
    Bates, T. R., & Lynch, J. P. (2001). Root hairs confer a competitive advantage under low phosphorus availability. Plant and Soil, 236, 243–250.CrossRefGoogle Scholar
  19. 19.
    Lynch, J. P., & Brown, K. M. (2001). Topsoil foraging—An architectural adaptation of plants to low phosphorus. Plant and Soil, 237, 225–237.CrossRefGoogle Scholar
  20. 20.
    Shen, H., Chen, J., Wang, Z., Yang, C., Sasaki, T., Yamamoto, Y., et al. (2006). Root plasma membrane H+-ATPase is involved in the adaptation of soybean to phosphorus starvation. Journal of Experimental Botany, 57, 1353–1362.CrossRefGoogle Scholar
  21. 21.
    Wang, X., Shen, J., & Liao, H. (2010). Acquisition or utilization, which is more critical for enhancing phosphorus efficiency in modern crops. Plant Science, 179, 302–306.CrossRefGoogle Scholar
  22. 22.
    Yang, H., Zhang, X., Gaxiola, R. A., Xu, G., Peer, W. A., & Murphy, A. S. (2014). Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit development under limiting phosphorus conditions. Journal of Experimental Botany, 65(12), 3045–3053.CrossRefGoogle Scholar
  23. 23.
    Guo, W., Zhao, J., Li, X., Qin, L., Yan, X., & Liao, H. (2011). A soybean β-expansin gene GmEXPB2 intrinsically involved in root system architecture responses to abiotic stresses. Plant Journal, 66(3), 541–552.CrossRefGoogle Scholar
  24. 24.
    Zhou, J., Xie, J., Liao, H., & Wang, X. (2013). Overexpression of β-expansin gene GmEXPB2 improves phosphorus efficiency in soyabean. Physiologia Plantarum, 150(2), 194–204.CrossRefGoogle Scholar
  25. 25.
    Wang, X., Wang, Y., Tian, J., Lim, B. L., Yan, X., & Liao, H. (2009). Overexpressing AtPAP15 enhances phosphorus efficiency in soyabean. Plant Physiology, 151(1), 233–240.CrossRefGoogle Scholar
  26. 26.
    Ma, X. F., Tudor, S., Butler, T., Ge, Y., Xi, Y., Bouton, J., et al. (2012). Transgenic expression of phytase and acid phosphatase genes in alfalfa (Medicago sativa) leads to improved phosphate uptake in natural soil. Molecular Breeding, 30, 377–391.CrossRefGoogle Scholar
  27. 27.
    Holme, I. B., Dionisio, G., Madsen, C. K., & Brinch-Pedersen, H. (2017). Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains. Plant Biotechnology Journal, 15, 415–422.CrossRefGoogle Scholar
  28. 28.
    Richardson, A. E., Barea, J. M., McNeill, A. M., & Prigent-Combaret, C. (2009). Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant and Soil, 321, 305–339.CrossRefGoogle Scholar
  29. 29.
    Richardson, A. E., Hadobas, P. A., & Hayes, J. E. (2001). Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant Journal, 25(6), 641–649.CrossRefGoogle Scholar
  30. 30.
    Gaxiola, R., Li, J., Undurraga, S., Dang, L., Allen, G., Alper, S., et al. (2001). Drought and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proceedings of the National Academy of Sciences of the United States of America, 98, 11444–11449.CrossRefGoogle Scholar
  31. 31.
    George, T. S., Richardson, A. E., Hadobas, P. A., & Simpson, R. J. (2004). Characterization of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: Growth and P nutrition in laboratory media and soil. Plant, Cell and Environment, 27, 1351–1361.CrossRefGoogle Scholar
  32. 32.
    Lung, S. C., Chan, W. L., Yip, W., Wang, L., Yeung, E. C., & Lim, B. L. (2005). Secretion of beta-propellar phytase from tobacco and Arabidopsis roots enhances phosphorus utilization. Plant Science, 169, 341–349.CrossRefGoogle Scholar
  33. 33.
    Liu, J. F., Zhao, C. Y., Ma, J., Zhang, G. Y., Li, M. G., Yan, G. J., et al. (2011). Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) with a fungal phytase gene improves phosphorus acquisition. Euphytica, 181(1), 31–40.CrossRefGoogle Scholar
  34. 34.
    Lung, S. C., & Lim, B. L. (2006). Assimilation of phytate-phosphorus by the extracellular phytase activity of tobacco (Nicotiana tabacum) is affected by the availability of soluble phytate. Plant and Soil, 279(1–2), 187–199.CrossRefGoogle Scholar
  35. 35.
    Curtis, T., & Halford, N. G. (2014). Food security: The challenge of increasing wheat yield and the importance of not compromising food safety. Annals of Applied Biology, 164(3), 354–372.CrossRefGoogle Scholar
  36. 36.
    Khan, I., & Zeb, A. (2007). Nutritional composition of Pakistani wheat varieties. Journal of Zhejiang University Science B, 8(8), 555–559.CrossRefGoogle Scholar
  37. 37.
    Centeno, C., Viveros, A., Brenes, A., Canales, R., Lozano, A., & de la Cuadra, C. (2001). Effect of several germination conditions on total P, phytate P, phytase, and acid phosphatase activities and inositol phosphate esters in rye and barley. Journal of Agricultural and Food Chemistry, 49, 3208–3215.CrossRefGoogle Scholar
  38. 38.
    George, T. S., Gregory, P. J., Hocking, P., & Richardson, A. E. (2008). Variation in root-associated phosphatase activities in wheat contributes to the utilization of organic P substrates in vitro, but does not explain differences in the P-nutrition of plants when grown in soils. Environmental and Experimental Botany, 64(3), 239–249.CrossRefGoogle Scholar
  39. 39.
    Mohsin, S., Malik, K. A., & Maqbool, A. (2015). Comparison of phytase activity in roots of wheat varieties grown under different phosphorus conditions. Research in Biotechnology, 6(3), 31–41.Google Scholar
  40. 40.
    Jones, H. D., Doherty, A., & Wu, H. (2005). Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods, 1, 5.CrossRefGoogle Scholar
  41. 41.
    Abid, N., Maqbool, A., & Malik, K. A. (2014). Screening commercial wheat (Triticum aestivum L.) varieties for Agrobacterium mediated transformation ability. Pakistan Journal of Agricultural Sciences, 51(1), 83–89.Google Scholar
  42. 42.
    Kang, T. J., & Yang, M. S. (2004). Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnology, 4, 20.CrossRefGoogle Scholar
  43. 43.
    Visarada, K. B. R. S., Saikishore, N., Kuriakose, S. V., Rani, V. S., Royer, M., Rao, S. V., et al. (2008). A simple model for selection and rapid advancement of transgenic progeny in sorghum. Plant Biotechnology Reports, 2, 47–58.CrossRefGoogle Scholar
  44. 44.
    Hoagland, D. R., & Arnon, D. I. (1950). The water-culture method for growing plants without soil. Circular - California Agricultural Experiment Station, 347, 1–32.Google Scholar
  45. 45.
    Hayes, J. E., Richardson, A. E., & Simpson, R. J. (1999). Phytase and acid phosphatase activities in extracts from roots of temperate pasture and legume seedlings. Australian Journal of Plant Physiology, 26, 801–809.CrossRefGoogle Scholar
  46. 46.
    Murphy, J., & Riley, J. P. (1962). A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta, 27, 31–36.CrossRefGoogle Scholar
  47. 47.
    Gunes, A., Inal, A., Alpaslan, M., & Cakmak, I. (2006). Genotypic variation in phosphorus efficiency between wheat cultivars grown under greenhouse and field conditions. Soil Science & Plant Nutrition, 52, 470–478.CrossRefGoogle Scholar
  48. 48.
    Das, A., Saha, D., & Mondal, T. K. (2013). An optimized method for extraction of RNA from tea roots for functional genomics analysis. Indian Journal of Biotechnology, 12, 129–132.Google Scholar
  49. 49.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402–408.CrossRefGoogle Scholar
  50. 50.
    Syers, J. K., Johnston, A. E., & Curtin, D. (2008). Efficiency of soil and fertilizer phosphorus use: Reconciling changing concepts of soil phosphorus behavior with agronomic information. Rome: Food and agriculture organization of the United Nations.Google Scholar
  51. 51.
    Tarafdar, J. C., & Jungk, A. (1987). Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biology and Fertility of Soils, 3(4), 199–204.CrossRefGoogle Scholar
  52. 52.
    Shenoy, V. V., & Kalagudi, G. M. (2005). Enhancing plant phosphorus use efficiency for sustainable cropping. Biotechnology Advances, 23, 501–513.CrossRefGoogle Scholar
  53. 53.
    Iti, G., Tantwai, K., Rajput, L. P. S., & Tiwari, S. (2012). Transgenic plants expressing phytase gene of microbial origin and their prospective application as feed. Food Technology and Biotechnology, 50(1), 3–10.Google Scholar
  54. 54.
    Pedersen, H. B., Hatzack, F., Stöger, E., Arcalis, E., Pontopidan, K., & Holm, P. B. (2006). Heat-stable phytases in transgenic wheat (Triticum aestivum L.): Deposition pattern, thermostability, and phytate hydrolysis. Journal of Agriculture and Food Chemistry, 54, 4624–4632.CrossRefGoogle Scholar
  55. 55.
    Hong, Y. F., Liu, C. Y., Cheng, K. J., Hour, A. L., Chan, M. T., Tseng, T. H., et al. (2008). The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato. Plant Molecular Biology, 67, 347–361.CrossRefGoogle Scholar
  56. 56.
    Pires, A. S., Cabral, M. G., Fevereiro, P., Stoger, E., & Abranches, R. (2008). High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures. Biotechnology Journal, 3, 916–923.CrossRefGoogle Scholar
  57. 57.
    Promdonkoy, P., Tang, K., Sornlake, W., Harnpicharnchai, P., Kobayashi, R. S., Ruanglek, V., et al. (2009). Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris. FEMS Microbiology Letters, 290(1), 18–24.CrossRefGoogle Scholar
  58. 58.
    Asmar, F. (1997). Variation in activity of root extracellular phytase between genotypes of barley. Plant and Soil, 195, 61–64.CrossRefGoogle Scholar
  59. 59.
    Zeller, S. L., Kalinina, O., Brunner, S., Keller, B., & Schmid, B. (2010). Transgene × environment interactions in genetically modified wheat. PLoS ONE, 5(7), e11405. doi:10.1371/journal.pone.0011405.CrossRefGoogle Scholar
  60. 60.
    Bruinsma, M., Kowalchuk, G. A., & Van-Veen, J. A. (2003). Effects of genetically modified plants on microbial communities and processes in soil. Biology and Fertility of Soils, 37, 329–337.Google Scholar
  61. 61.
    Castaldini, M., Turrini, A., Sbrana, C., et al. (2005). Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms. Applied and Environmental Microbiology, 71, 6719–6729.CrossRefGoogle Scholar
  62. 62.
    Griffiths, B. S., Caul, S., Thompson, J., Birch, A. N. E., Scrimegour, C., Cortet, J., et al. (2006). Soil microbial and faunal community responses to Bt maize and insecticide in two soils. Journal of Environmental Quality, 35, 734–741.CrossRefGoogle Scholar
  63. 63.
    Philippot, L., Kuffner, M., Cheneby, D., Depret, G., Lauguerre, G., & Martin-Laurent, F. (2006). Genetic structure and activity of the nitrate reducers community in the rhizosphere of different cultivars of maize. Plant and Soil, 287, 177–186.CrossRefGoogle Scholar
  64. 64.
    Lupwayi, N. Z., Hanson, K. G., Harker, K. N., Clayton, G. W., Blackshaw, R. E., O’Donovan, J. T., et al. (2007). Soil Microbial biomass, functional diversity and enzyme activity in glyphosate-resistant wheat–canola rotations under low-disturbance direct seeding and conventional tillage. Soil Biology & Biochemistry, 39, 1418–1427.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Samreen Mohsin
    • 1
  • Asma Maqbool
    • 1
  • Mehwish Ashraf
    • 1
  • Kauser Abdulla Malik
    • 1
  1. 1.Department of Biological SciencesForman Christian College (A Chartered University)LahorePakistan

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