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