Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs
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.
KeywordsBarley 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.
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.
- 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
- 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
- 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
- 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
- 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
- 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
- Erlang-Nielsen M (2014) Plant nutrition and health. Dissertation, Aarhus UniversityGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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