Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing
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Background and Aims: Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. Methods: Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. Key Results: Mutagenesis efficiency among T0 regenerated plantlets was 50–65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. Conclusions: Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.
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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 Zeng et al., 2020
Transgene-free homozygous mutants are desirable and in barley are feasible in the T1 generation.
“Transgene-free homozygous mutants are desirable and in barley are feasible in the T1 generation (Zeng et al., 2020).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Zeng et al., 2020
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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
- Zeng, Z., Han, N., Liu, C., Buerte, B., Zhou, C., Chen, J., Wang, M., Zhang, Y., Tang, Y., Zhu, M., Wang, J., Yang, Y., & Bian, H.
- Affiliations
- Zhejiang University (CN)
- Zhejiang University of Technology (CN)
- Tibet Academy of Agricultural and Animal Husbandry Sciences (CN)
- Pennsylvania State University (US)
- Corresponding author
- Bian, Hongwu
- ORCID
- Venue
- Annals of Botany
- Publisher
- Oxford University Press (OUP)
- Published
- 2020-06-24
- Volume
- 126
- Issue
- 5
- Pages
- 929-942
- ISSN
- 0305-7364 (ISSN-L)
- 1095-8290
- PMC
- PMC7539355
- OpenAlex
- W3037109705
- Cited by
- 86
- References
- 47
- Citations by year
- 2026: 10 · 2025: 20 · 2024: 13 · 2023: 19 · 2022: 12 · 2021: 9 · 2020: 3
- FWCI
- 3.57
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Annals of Botany (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Natural Science Foundation of China (31971932, 31771776)
- China Agriculture Research System
- Science Foundation of Zhejiang Province (LGN18C130001)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- barley, vitamin E biosynthesis, tocotrienols, tocopherols, HPT, tocopherol biosynthesis, knockout mutants, CRISPR/Cas9 genome editing, grain starch content, grain size
- MeSH terms
- Gene Editing, Hordeum, Humans, Vitamin E, Tocopherols, Tocotrienols, CRISPR-Cas Systems
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