Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs
Abstract
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.
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In barley, CRISPR/Cas9 gene editing has been successfully used to create insertion/deletion (InDel) mutations in eIF4E, HvMPK6, HvNud, HvPAPhy, HvPTST1, HvGBSS1, HvHPT, and HvHGGT genes to improve desirable traits.
“In barley, CRISPR/Cas9 gene editing has been successfully used to create insertion/deletion (InDel) mutations in eukaryotic translation initiation factor 4E (elF4E), H. vulgare mitogen-activated protein kinase 6 (HvMPK6), H. vulgare nudum (HvNud), H. vulgare purple acid phosphatase phytase (HvPAPhy), H. vulgare protein targeting to starch 1 (HvPTST1), H. vulgare granule-bound starch synthase 1 (Hv”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Holme et al., 2017
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“…1 ( HvGBSS1 ), H. vulgare homogentisate phytyltransferase ( HvHPT ), and H. vulgare homogentisate geranylgeranyl trans‐ ferase ( HvHGGT ) genes to improve desirable traits (Holme et al., 2017; Zhong et al., 2019; Zeng et al., 2020; Křenek et al., 2021; Kershanskaya et al., 2022; Zang et al., 2022).”
- Authors
- Holme, I. B., Wendt, T., Gil-Humanes, J., Deleuran, L. C., Starker, C. G., Voytas, D. F., & Brinch-Pedersen, H.
- Affiliations
- Aarhus University (DK)
- University of Minnesota (US)
- Corresponding author
- Holme, Inger B.
- Venue
- Plant Molecular Biology
- Published
- 2017-07-28
- Volume
- 95
- Issue
- 1-2
- Pages
- 111-121
- ISSN
- 0167-4412 (ISSN-L)
- 1573-5028
- OpenAlex
- W2741798010
- Cited by
- 71
- References
- 53
- Citations by year
- 2026: 2 · 2025: 8 · 2024: 5 · 2023: 10 · 2022: 9 · 2021: 5 · 2020: 15 · 2019: 11 · 2018: 6
- FWCI
- 8.61
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Plant Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Danish Ministry of Food, Agriculture and Fisheries (3304-FVFP-09-B-006)
- Brd. Hartmann’s foundation (A27246)
- National Science Foundation (IOS-1444511, IOS-1339209)
- Topics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Phytase and its Applications
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant nutrient uptake and metabolism
- Keywords
- cis-regulatory elements, grain filling, seed germination, phosphorus availability, gene duplication, CRISPR/Cas9 mutagenesis, TALEN mutagenesis
- MeSH terms
- Transcription Activator-Like Effector Nucleases, Hordeum, Base Sequence, DNA, Bacterial, Genetic Vectors, Homozygote, Mutation, Oxygen Consumption, 6-Phytase, Seeds, Sequence Alignment, Germination, CRISPR-Cas Systems
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