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

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Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs. / Holme, Inger B; Wendt, Toni; Gil-Humanes, Javier; Deleuran, Lise C; Starker, Colby G; Voytas, Daniel F; Brinch-Pedersen, Henrik.

In: Plant Molecular Biology, Vol. 95, No. 1-2, 09.2017, p. 111-121.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Holme, IB, Wendt, T, Gil-Humanes, J, Deleuran, LC, Starker, CG, Voytas, DF & Brinch-Pedersen, H 2017, 'Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs', Plant Molecular Biology, vol. 95, no. 1-2, pp. 111-121. https://doi.org/10.1007/s11103-017-0640-6

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Holme IB, Wendt T, Gil-Humanes J, Deleuran LC, Starker CG, Voytas DF et al. Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs. Plant Molecular Biology. 2017 Sep;95(1-2):111-121. https://doi.org/10.1007/s11103-017-0640-6

Author

Holme, Inger B ; Wendt, Toni ; Gil-Humanes, Javier ; Deleuran, Lise C ; Starker, Colby G ; Voytas, Daniel F ; Brinch-Pedersen, Henrik. / Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs. In: Plant Molecular Biology. 2017 ; Vol. 95, No. 1-2. pp. 111-121.

Bibtex

@article{3019ec101a38459ca3427bc0c24bcf14,
title = "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.",
keywords = "Barley, CRISPR/Cas9, GCN4/Skn1/RY motif, HvPAPhy_a, Mature grain phytase activity, TALENs",
author = "Holme, {Inger B} and Toni Wendt and Javier Gil-Humanes and Deleuran, {Lise C} and Starker, {Colby G} and Voytas, {Daniel F} and Henrik Brinch-Pedersen",
year = "2017",
month = "9",
doi = "10.1007/s11103-017-0640-6",
language = "English",
volume = "95",
pages = "111--121",
journal = "Plant Molecular Biology",
issn = "0167-4412",
publisher = "Springer Netherlands",
number = "1-2",

}

RIS

TY - JOUR

T1 - Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs

AU - Holme, Inger B

AU - Wendt, Toni

AU - Gil-Humanes, Javier

AU - Deleuran, Lise C

AU - Starker, Colby G

AU - Voytas, Daniel F

AU - Brinch-Pedersen, Henrik

PY - 2017/9

Y1 - 2017/9

N2 - 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.

AB - 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.

KW - Barley

KW - CRISPR/Cas9

KW - GCN4/Skn1/RY motif

KW - HvPAPhy_a

KW - Mature grain phytase activity

KW - TALENs

U2 - 10.1007/s11103-017-0640-6

DO - 10.1007/s11103-017-0640-6

M3 - Journal article

VL - 95

SP - 111

EP - 121

JO - Plant Molecular Biology

JF - Plant Molecular Biology

SN - 0167-4412

IS - 1-2

ER -