Plant Molecular Biology

, Volume 95, Issue 1–2, pp 111–121 | Cite as

Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs

  • Inger B. Holme
  • Toni Wendt
  • Javier Gil-Humanes
  • Lise C. Deleuran
  • Colby G. Starker
  • Daniel F. Voytas
  • Henrik Brinch-Pedersen


In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA. Additionally, the analysis of the HvPAPhy_a promoter region containing the GCN4/Skn1/RY motif highlights its importance for HvPAPhy_a expression as the MGPA in grains of plant lines with mutations within this motif is significantly reduced. Interestingly, lines with deletions located downstream of the motif show even lower MGPA levels, indicating that the GCN4/SKn1/RY motif is not the only element responsible for the level of PAPhy_a expression during grain maturation. Mutant grains with very low MPGA showed delayed germination as compared to grains of wild type barley. As grains with high levels of preformed phytases would provide more readily available phosphorous needed for a fast germination, this indicates that faster germination may be implicated in the positive selection of the ancient PAPhy gene duplication that lead to the creation of the PAPhy_a gene.


Barley CRISPR/Cas9 GCN4/Skn1/RY motif HvPAPhy_a Mature grain phytase activity TALENs 



The authors thank Rikke B Jacobsen, Lis B Holte and Ole B Hansen for skilful technical assistance. The research was funded by a grant to IBH from the Danish Ministry of Food, Agriculture and Fisheries (3304-FVFP-09-B-006), a Grant to IBH from Brd. Hartmann’s foundation (A27246) and Grants to DFV from the National Science Foundation (IOS-1444511 and IOS-1339209). Javier Gil-Humanes acknowledges the Fundación Alfonso Martin Escudero for his post-doctoral fellowship.

Author contributions

IBH, TW, JG-H and LCD all contributed to the experimental part of this research. CGS, DFV and HB-P advised on the experimental part. IBH and HB-P wrote the initial draft for the paper and the initial draft was carefully revised by JG-H, TW, CGS, LCD and DFV.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2017_640_MOESM1_ESM.pdf (1 mb)
Supplementary material 1 (PDF 1031 KB)
11103_2017_640_MOESM2_ESM.pdf (390 kb)
Supplementary material 2 (PDF 390 KB)
11103_2017_640_MOESM3_ESM.pdf (335 kb)
Supplementary material 3 (PDF 335 KB)


  1. Baumlein H, Nagy I, Villarroel R, Inze D, Wobus U (1992) Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene. Plant J 2:233–239. doi: 10.1046/j.1365-313X.1992.t01-45-00999.x PubMedGoogle Scholar
  2. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designer zinc finger nucleases. Science 300:764. doi: 10.1126/science.1079512 CrossRefPubMedGoogle Scholar
  3. Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846. doi: 10.1126/science.1204094 CrossRefPubMedGoogle Scholar
  4. Bradford KJ, Bello P, Fu JC, Barros M (2013) Single-seed respiration: a new method to assess seed quality. Seed Sci Technol 41:420–438. doi: 10.15258/sst.2013.41.3.09 CrossRefGoogle Scholar
  5. 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. doi: 10.1023/A:1009690730620 CrossRefGoogle Scholar
  6. Brinch-Pedersen H, Sørensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125. doi: 10.1016/S1360-1385(01)02222-1 CrossRefPubMedGoogle Scholar
  7. Brinch-Pedersen H, Madsen CK, Holme IB, Dionisio G (2014) Increased understanding of the cereal phytase complement for better mineral bio-availability and resource management. J Cereal Sci 59:373–381. doi: 10.1016/j.jcs.2013.10.003 CrossRefGoogle Scholar
  8. Char SN, Unger-Wallace E, Frame B, Briggs SA, Main M, Spalding MH, Vollbrecht E, Wang K, Yang B (2015) Heritable site-specific mutagenesis using TALENs in maize. Plant Biotechnol J 13:1002–1010. doi: 10.1111/pbi.12344 CrossRefPubMedGoogle Scholar
  9. Christian ML, Demorest ZL, Starker CG, Osborn MJ, Nyquist MD, Zhang Y, Carlson DF, Bradley P, Bogdanove AJ, Voytas DF (2012) Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues. PLoS ONE 7(9):e45383. doi: 10.1371/journal.pone.0045383 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Clasen BM, Stoddard TJ, Luo S, Demorest ZL, Li J, Cedrone F, Tibebu R, Davison S, Ray EE, Daulhac A, Coffman A, Yabandith A, Retterath A, Haun W, Baltes NJ, Mathis L, Voytas DF, Zhang F (2016) Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnol J 14:169–176. doi: 10.1111/pbi.12370 CrossRefPubMedGoogle Scholar
  11. Dionisio G, Holm PB, Brinch-Pedersen H (2007) Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) multiple inositol polyphosphate phosphatases (MINPPs) are phytases expressed during grain filling and germination. Plant Biotechnol J 5:325–338. doi: 10.1111/j.1467-7652.2007.00244.x CrossRefPubMedGoogle Scholar
  12. Dionisio G, Madsen CK, Holm PB, Welinder KG, Jørgensen M, Stroger E, Arcalis E, Brinch Pedersen H (2011) Cloning and characterization of purple acid phoshatase phytases from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), maize (Zea maize L.) and rice (Oryza sativa L.). Plant Physiol 156:1087–1100. doi: 10.1104/pp.110.164756 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Duan Y, Li J, Qin R, Xu R, Li H, Yang Y, Ma H, Li L, Wei P, Yang J (2016) Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis. Plant Mol Biol 90:49–62. doi: 10.1007/s11103-015-0393-z CrossRefPubMedGoogle Scholar
  14. Engelen AJ, van der Heeft FC, Randsdorp PH, Smit ELC (1994) Simple and rapid-determination of phytase activity. J AOAC Int 77:760–764PubMedGoogle Scholar
  15. Epinat JC, Arnould S, Chames P, Rochaix P, Desfontaines D, Puzin C, Patin A, Zanghellini A, Pâques F, Lacroix E (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucl Acids Res 31:2952–2962. doi: 10.1093/nar/gkg375 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Erlang-Nielsen M (2014) Plant nutrition and health. Dissertation, Aarhus UniversityGoogle Scholar
  17. Fauteux F, Stromvik M (2009) Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae. BMC Plant Biol 9:126. doi: 10.1186/1471-2229-9-126 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Forster C, Arthur E, Crespi S, Hobbs SLA, Mullineaux P, Casey R (1994) Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco. Plant Mol Biol 26:235–248. doi: 10.1007/BF00039535 CrossRefPubMedGoogle Scholar
  19. Fujiwara T, Beachy RN (1994) Tissue-specific and temporal regulation of a β-conglycinin gene: roles of the RY repeat and other cis-acting elements. Plant Mol Biol 24:261–272. doi: 10.1007/BF00020166 CrossRefPubMedGoogle Scholar
  20. Grant CA, Flaten DN, Tomasiewicz DJ, Sheppard SC (2001) The importance of early season phosphorus nutrition. Can J Plant Sci 81:211–224. doi: 10.4141/P00-093 CrossRefGoogle Scholar
  21. Greiner R, Jany KD, Larsson MA (2000) Identification and properties of myo-inositol hexakisphosphate phosphohydrolases (phytases) from barley (Hordeum vulgare). J Cereal Sci 31:127–139. doi: 10.1006/jcrs.1999.0254 CrossRefGoogle Scholar
  22. Gurushidze M, Hensel G, Hiekel S, Schedel S, Valkov V, Kumlehn J (2014) True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells. PLoS ONE 9(3):e92046. doi: 10.1371/journal.pone.0092046 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas DF, Zhang F (2014) Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J 12:934–940. doi: 10.1111/pbi.12201 CrossRefPubMedGoogle Scholar
  24. Holme IB, Dionisio G, Brinch-Pedersen H, Wendt T, Madsen CK, Vincze E, Holm PB (2012) Cisgenic barley with improved phytase activity. Plant Biotechnol J 10:237–247. doi: 10.1111/j.1467-7652.2011.00660.x CrossRefPubMedGoogle Scholar
  25. Jia H, Orbovic V, Jones JB, Wang N (2016) Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating Xcc∆pthA4:dCsLOB1.3 infection. Plant Biotechnol J 14:1291–1301. doi: 10.1111/pbi.12495 CrossRefPubMedGoogle Scholar
  26. Jiang WZ, Henry IM, Lynagh PG, Cormai L, Cahoon EB, Weeks DP (2016) Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnol J 15:648–657. doi: 10.1111/pbi.12663 CrossRefGoogle Scholar
  27. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. doi: 10.1126/science.1225829 CrossRefPubMedGoogle Scholar
  28. Juhasz A, Makai S, Sebestyen E, Tamas L, Balazs E (2011) Role of conserved non-coding regulatory elements in LMW glutenin gene expression. PLoS ONE 6(12):e29501. doi: 10.1371/journal.pone.0029501 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kapusi E, Corcuera-Gomez M, Melnik S, Stoger E (2017) Heritable genomic fragment deletions and small indels in the putative ENGase gene induced by CRISPR/Cas9 in barley. Front Plant Sci 8:540. doi: 10.3389/fpls.2017.00540 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Karimi M, Inzé D, Depicker A (2002) Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195. doi: 10.1016/S1360-1385(02)02251-3 CrossRefPubMedGoogle Scholar
  31. Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. doi: 10.1186/s13059-015-0826-7 PubMedPubMedCentralGoogle Scholar
  32. Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotech 30:390–392. doi: 10.1038/nbt.2199 CrossRefGoogle Scholar
  33. Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B., Yang Z., Li H., Lin Y., Xie Y., Shen R., Chen S., Wang Z., Chen Y., Guo J., Chen L., Zhao X., Dong Z., and Liu Y.-G. (2015). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8, 1274–1284. doi: 10.1016/j.molp.2015.04.007 CrossRefPubMedGoogle Scholar
  34. Madsen CK, Dionisio G, Holme IB, Holm PB, Brinch-Pedersen H (2013) High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene. J Exp Bot 64:3111–3123. doi: 10.1093/jxb/ert116 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Mann DGJ, LaFayette PR, Abercrombie LL, King ZR, Mazarei M, Halter MC, Poovaiah CR, Baxter H, Shen H, Dixon RA, Parrott WA, Stewart Jr CN (2012) Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.). Plant Biotechnol J 10: 226–236. doi: 10.1111/j.1467-7652.2011.00658.x CrossRefPubMedGoogle Scholar
  36. Muller M, Knudsen S (1993) The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant J 4:343–355. doi: 10.1046/j.1365-313X.1993.04020343.x CrossRefPubMedGoogle Scholar
  37. Nakano T, John T, Tokumoto E, Hayakawa T (1999). Purification and characterization of phytase from wheat bran of Triticum aestivum L. cv. Nourin #61. Food Sci Technol Res 5:18–23.CrossRefGoogle Scholar
  38. Onate L, Vicente-Carbajosa J, Lara P, Diaz I, Carbonero P (1999) Barley BLZ2, a seed-specific bZIP protein that interacts with BLZ1 in vivo and activates transcription from the GCN4-like motif of B-hordein promoters in barley endosperm. J Biol Chem 274:9175–9182. doi: 10.1074/jbc.274.14.9175 CrossRefPubMedGoogle Scholar
  39. Ranford JC, Bryce JH, Morris PC (2002) PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy. J Exp Bot 53:147–148. doi: 10.1093/jexbot/53.366.147 PubMedGoogle Scholar
  40. Reidt W, Wohlfarth T, Ellerstrom M, Czihal A, Tewes A, Ezcurra I, Rask L, Baumlein H (2000) Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product. Plant J 21:401–408. doi: 10.1046/j.1365-313x.2000.00686.x CrossRefPubMedGoogle Scholar
  41. Robinson WD, Carson I, Ying S, Ellis K, Plaxton WC (2012) Eliminating the purple acid phosphatase ATPAP26 in Arabidopsis thaliana delays leaf senescence and impairs phosphorus remolilization. New Phytol 196:1024–1029. doi: 10.1111/npi.12006 CrossRefPubMedGoogle Scholar
  42. Shan Q, Zhang Y, Chen K, Zhang K, Gao C (2015) Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnol J 13:791–800. doi: 10.1111/pbi.12312 CrossRefPubMedGoogle Scholar
  43. Vain P, Afolabi AS, Worland B, Snape JW (2003) Transgene behavior in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107:210–217. doi: 10.1007/s00122-003-1255-7 CrossRefPubMedGoogle Scholar
  44. Voytas DF (2016) Editorial prerogative and the plant genome. J Genet Genom 43:229–232. doi: 10.1016/j.jgg.2016.03.004 CrossRefGoogle Scholar
  45. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951. doi: 10.1038/nbt.2969 CrossRefPubMedGoogle Scholar
  46. Weeks DP, Spalding MH, Yang B (2016) Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol J 14:483–495. doi: 10.1111/pbi.12448 CrossRefPubMedGoogle Scholar
  47. Wendt T, Holm PB, Starker CG, Christian M, Voytas DF, Brinch-Pedersen H, Holme IB (2013) TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants. Plant Mol Biol 83:279–285. doi: 10.1007/s11103-013-0078-4 CrossRefPubMedGoogle Scholar
  48. White PJ, Veneklaas EJ (2012) Nature and nurture: the importance of seed phosphorus content. Plant Soil 357:1–8. doi: 10.1007/s11104-012-1128-4 CrossRefGoogle Scholar
  49. Wu C, Suzuki A, Washida H, Takaiwa F (1998) The GCN4 motif in a rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic rice plants. Plant J 14:673–683. doi: 10.1046/j.1365-313x.1998.00167.x CrossRefPubMedGoogle Scholar
  50. Wu C, Washida H, Onodera Y, Harada K, Takaiwa F (2000) Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutenin gene promoter: minimal cis-element requirements for endosperm-specific gene expression. Plant J 23:415–421. doi: 10.1046/j.1365-313x.2000.00797.x CrossRefPubMedGoogle Scholar
  51. Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20–27. doi: 10.1104/pp.112.20517 CrossRefPubMedGoogle Scholar
  52. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu K-J (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807. doi: 10.1111/pbi.12200 CrossRefPubMedGoogle Scholar
  53. Zhang D, Zhenxiang L, Li J (2016a) Targeted gene manipulation in plants using CRISPR/Cas technology. J Genet Genom 43:251–262. doi: 10.1016/j.jgg.2016.03.001 CrossRefGoogle Scholar
  54. Zhang H, Gou F, Zhang J, Liu W, Li Q, Mao Y, Botella JR, Zhu J (2016b) TALEN-mediated targeted mutagenesis produces a large variety of heritable mutations in rice. Plant Biotechnol J 14:186–194. doi: 10.1111/pbi.12372 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of Molecular Biology and Genetics, Research Centre FlakkebjergAarhus UniversitySlagelseDenmark
  2. 2.Department of Genetics, Cell Biology and Development and Center for Genome EngineeringUniversity of MinnesotaMinneapolisUSA
  3. 3.Calyxt Inc.New BrightonUSA
  4. 4.Department of Agroecology, Research Centre FlakkebjergAarhus UniversitySlagelseDenmark

Personalised recommendations