Journal of Plant Biochemistry and Biotechnology

, Volume 28, Issue 4, pp 470–482 | Cite as

Genetically engineered rice with appA gene enhanced phosphorus and minerals

  • Sananda Bhattacharya
  • Shinjini Sengupta
  • Aritra Karmakar
  • Sailendra Nath Sarkar
  • Gaurab Gangopadhyay
  • Karabi Datta
  • Swapan Kumar DattaEmail author
Original Article


Phytic acid is the major source of phosphorus and other mineral bound compounds in many plant tissues especially seeds and bran of cereals. During germination, phytase enzyme degrades phytic acid and bound phosphate and minerals are released. The monogastric animal cannot digest phytate due to lack of the phytase enzyme. Considering that, we have generated low phytate rice by over expressing appA gene cloned from E. coli under the aleurone-specific promoter of maize zein gene. Molecular analysis confirmed the stable integration of transgene and plants were grown up to T3 generation. The T3 seeds showed around 45% decrease in seed phytate content with a fourfold increase of inorganic phosphorus (Pi) level. The enhanced iron and zinc was twofold and threefold respectively in polished seeds of transgenic plants. There was no change in germination behaviour and other morphological traits in transgenic seeds. Thus, this result provides evidence that tissue-specific expression of bacterial phytase can lead to the reduction of phytic acid in rice seeds without hampering its other physiological processes and phenotypic cost.


appA phytase Mineral fortification Phytic acid Transgenic rice 



Apoplastic periplasmic protein A


Inositol mono phosphatase


Inositol 1,3,4 trisphosphate 5/6 kinase


Inositol 1,3,4,5,6 pentakisphosphate 2-kinase


Myo-inositol phosphate synthase


Low phytic acid



We are thankful to ICAR (Sanction No. DRR/CRP/Biofortification/2015) and DBT, (Sanction number: BT/PR12656/COE/34/22/2015) for the financial support. Ms. Sayani Majumdar for laboratory assistance, Mr. Pratap Ghosh and Mr. Sujoy Mondal for greenhouse work are duly acknowledged. We also acknowledge Mr. Rahul Bezbaruah, Scientific Officer (Chemical), National Test House, Govt. of India, Kolkata, for his assistance in analysis of myo-inositol through GC–MS.

Author contributions

SB designed and performed the experiments, wrote manuscript, prepared figures and tables. SSG and AK helped PCR screening. SNS guided in AAS experiment and helped in manuscript editing. GG helped in analysing the data, guided in experiments and helped in manuscript preparation. SKD and KD designed and supervised all the experiments, provided materials and revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2019_505_MOESM1_ESM.docx (5.1 mb)
Supplementary material 1 (DOCX 5180 kb)


  1. Ali N, Paul S, Gayen D, Sarkar SN, Datta SK, Datta K (2013a) Development of low phytate rice by RNAi mediated seed-specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene (IPK1). PLoS ONE 8(7):e68161PubMedPubMedCentralGoogle Scholar
  2. Ali N, Paul S, Gayen D, Sarkar SN, Datta SK, Datta K (2013b) RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice. Rice 6:12PubMedPubMedCentralGoogle Scholar
  3. Bilyeu KD, Zeng P, Coello P, Zhang ZJ, Krishnan HB, Bailey A, Beuselinck PR, Polacco JC (2008) Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase. Plant Physiol 146:468–477PubMedPubMedCentralGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  5. Bregitzer P, Raboy V (2006) Effects of four independent low-phytate mutations on barley agronomic performance. Crop Sci 46:1318–1322Google Scholar
  6. Chappell AS, Scaboo AM, Wu X, Nguyen H, Pantalone VR, Bilyeu KD (2006) Characterization of the MIPS gene family in Glycine max. Plant Breed 125:493–500Google Scholar
  7. Chen PS, Toribara TY, Warner H (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758Google Scholar
  8. Chhetri DR, Mukherjee AK, Adhikari J (2006) Myo-inositol content in pteridophytes and the isolation and characterization of l-myo-inositol-1-phosphate synthase from Diplopterygium glaucum Braz. J Plant Physiol 18:291–298Google Scholar
  9. Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, Datta SK (1998) Constitutive and tissue specific differential expression of cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theor Appl Genet 97:20–30Google Scholar
  10. Datta K, Koukolikova-Nicola Z, Baisakh N, Oliva N, Datta SK (2000) Agrobacterium mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor Appl Genet 100:832–839Google Scholar
  11. Doria E, Galleschi L, Calucci L, Pinzino C, Pilu R, Cassani E, Nielsen E (2009) Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant. J Exp Bot 60:967–978PubMedGoogle Scholar
  12. Feng X, Yoshida KT (2004) Molecular approaches for producing low-phytic-acid grains in rice. Plant Biotechnol 21:183–189Google Scholar
  13. Gayen D, Sarkar SN, Datta SK, Datta K (2013) Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart. Food Chem 138:733–2070Google Scholar
  14. Giovinazzo G, Manzocchi LA, Bianchi MW, Coraggio I, Viotti A (1992) Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts. Plant Mol Biol 19:257–263PubMedGoogle Scholar
  15. Greiner R, Konietzny U, Jany KD (1993) Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys 303:107–113PubMedGoogle Scholar
  16. Gupta RK, Gangoliya SS, Singh NK (2015) Reduction of phytic acid and enhancement of bioavailable micronutrients in food grains. J Food Sci Technol 52(2):676–684PubMedGoogle Scholar
  17. Guttieri MJ, Peterson KM, Souza EJ (2006) Agronomic performance of low phytic acid wheat. Crop Sci 46:2623–2629Google Scholar
  18. Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF (2004) Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 13:29–39PubMedGoogle Scholar
  19. Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G (1997) Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR. Theor Appl Genet 95:313–320Google Scholar
  20. IRRI (2002) Standard evaluation system for rice (SES). International Rice Research Institute, ManilaGoogle Scholar
  21. Jacob L (1989) High-Performance Liquid Chromatography analysis of phytic acid on a pH stable, macroporous polymer column. Cereal Chem 66:510–515Google Scholar
  22. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-Glucuronidase is a sensitive and versatile fusion marker in higher plants. EMBO J 6:3901–3907PubMedPubMedCentralGoogle Scholar
  23. Kim SI, Tai TH (2011) Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Mol Genet Genomics 286:119–133PubMedGoogle Scholar
  24. Kim S, Tai T (2014) Identification of novel rice low phytic acid mutations via TILLING by sequencing. Mol Breed 34:1717–1729Google Scholar
  25. Kuwano M, Ohyama A, Tanaka Y, Mimura T, Takaiwa F, Yoshida KT (2006) Molecular breeding for transgenic rice with low phytic acid phenotype through manipulating myo inositol 3 phosphate synthase gene. Mol Breed 18:263–272Google Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Method 25:402–408Google Scholar
  27. Loewus FA, Murthy PPN (2000) Myo-inositol metabolism in plants. Plant Sci 150:1–19Google Scholar
  28. Lucca P, Hurrell R, Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392–397Google Scholar
  29. Majumder AL, Chatterjee A, Ghosh Dastidar K, Majee M (2003) Diversification and evolution of l-myo-inositol 1-phosphate synthase. FEBS Lett 553:3–10PubMedGoogle Scholar
  30. Mei C, Wassom JJ, Wildholm JM (2004) Expression specificity of the Globulin-1 promoter driven transgene (chitanase) in maize seed tissue. Maydica 49:255–265Google Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  32. Nunes ACS, Vianna GR, Cuneo F, Amaya-Farfan J, de Capdeville G, Rech EL, Aragao FJL (2006) RNAi mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content. Planta 224:125–132PubMedGoogle Scholar
  33. Ogawa M, Tanaka K, Kasai Z (1979) Phytic acid formation in dissected ripening rice grains. Agric Biol Chem 43:2211–2213Google Scholar
  34. Panzeri D, Cassani E, Doria E, Tagliabue G, Forti L et al (2011) A defective ABC transporter of the MRP family, responsible for the bean lpa1 mutation, affects the regulation of the phytic acid pathway, reduces seed myo-inositol and alters ABA sensitivity. New Phytol 191:70–83PubMedGoogle Scholar
  35. Paul S, Ali N, Gayen D, Datta SK, Datta K (2012) Molecular breeding of Osfer2 gene to increase iron nutrition in rice grain. GM Crops Food 3:310–316PubMedGoogle Scholar
  36. Qiao-quan L, Qian-feng L, Li J, Da-jiang Z, Hong-mei W, Ming-hong G, Quan-hong Y (2006) Transgenic expression of the recombinant phytase in rice (Oryza sativa). Rice Sci 13:2Google Scholar
  37. Raboy V (2009) Approaches and challenges to engineering seed phytate and total phosphorus. Plant Sci 177:281–296Google Scholar
  38. Raboy V, Gerbasi PF, Young KA, Stoneberg SD, Pickett SG, Bauman AT, Murthy PP, Sheridan WF, Ertl DS (2000) Origin and seed phenotype of maize low phytic acid 1–1 and low phytic acid 2–1. Plant Physiol 124:355–368PubMedPubMedCentralGoogle Scholar
  39. Sambrook JF, Russell DW (eds) (2001) Molecular cloning: A laboratory manual, 3rd edn., vol 1, 2, 3. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  40. Shi J, Wang H, Schellin K, Li B, Faller M, Stoop JM, Meeley RB, Ertl DS, Ranch JP, Glassman K (2007) Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds. Nat Biotechnol 25:930–937PubMedGoogle Scholar
  41. Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L, Krishnan S, Oliveira M, Goto F, Datta SK (2003) Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Sci 164:371–378Google Scholar
  42. Walker DR, Scaboo AM, Pantalone VR, Wilcox JR, Boerma HR (2006) Genetic mapping of loci associated with seed phytic acid content in CX1834-1-2 soybean. Crop Sci 46:390–397Google Scholar
  43. Yang Y, Dai L, Xia H, Zhu K, Liu H, Chen K (2013) Protein profile of rice (Oryza sativa) seeds. Genet Mol Biol 36(1):87–92PubMedPubMedCentralGoogle Scholar
  44. Yoshida K, Wada T, Koyama H, Mizobuchi-Fukuoka R, Naito S (1999) Temporal and spatial patterns of accumulation of the transcript of myo-inositol-1-phosphate synthase and phytin-containing particles during seed development in rice. Plant Physiol 119:65–72PubMedPubMedCentralGoogle Scholar
  45. Zuo-ping W, Li-hua D, Lü-shui W, Xiang-yang D, Xi-qin F, Ye-yun X, Guo-ying X (2017) Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity. J Integr Agric 16(4):774–788Google Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  1. 1.Department of Botany, Annex Building IIUniversity of CalcuttaKolkataIndia
  2. 2.Division of Plant BiologyBose Institute (Main Campus)KolkataIndia

Personalised recommendations