CRISPR/Cas9 gene editing and natural variation analysis demonstrate the potential for HvARE1 in improvement of nitrogen use efficiency in barley
Abstract
Nitrogen is a major determinant of grain yield and quality. As excessive use of nitrogen fertilizer leads to environmental pollution and high production costs, improving nitrogen use efficiency (NUE) is fundamental for a sustainable agriculture. Here, we dissected the role of the barley abnormal cytokinin response1 repressor 1 ( HvARE1 ) gene, a candidate for involvement in NUE previously identified in a genome‐wide association study, through natural variation analysis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated gene editing. HvARE1 was predominantly expressed in leaves and shoots, with very low expression in roots under low nitrogen conditions. Agrobacterium ‐mediated genetic transformation of immature embryos ( cv . Golden Promise) with single guide RNAs targeting HvARE1 generated 22 T 0 plants, from which four T 1 lines harbored missense and/or frameshift mutations based on genotyping. Mutant are1 lines exhibited an increase in plant height, tiller number, grain protein content, and yield. Moreover, we observed a 1.5‐ to 2.8‐fold increase in total chlorophyll content in the flag leaf at the grain filling stage. Delayed senescence by 10–14 d was also observed in mutant lines. Barley are1 mutants had high nitrogen content in shoots under low nitrogen conditions. These findings demonstrate the potential of ARE1 in NUE improvement in barley.
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CRISPR/Cas9 is a robust and versatile tool for precise mutation creation and trait improvement.
“In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a robust and versatile tool for precise mutation creation and trait improvement.”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Several barley genes have been exploited to address different stress conditions, including HVA1 for drought, HKT1;1 for salinity, and ARE1 for nitrogen use efficiency.
“Several barley genes have been exploited to address different stress conditions (Zhou et al., 2016;Hazzouri et al., 2018;Karunarathne et al., 2020;Mwando et al., 2020), including Hordeum vulgare aleurone 1 (HVA1) for drought, high-affinity K + transporter 1;1 (HKT1;1) for salinity, and abnormal cytokinin response 1 repressor 1 (ARE1) for nitrogen use efficiency (NUE) (Sivamani et al., 2000;Han et”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
More precise gene-editing technologies such as CRISPR/Cas9 gene editing, base editing, and prime editing are imperative to develop new barley lines with superior qualities and better performance under stress conditions.
“Therefore, more precise gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) gene editing, base editing, and prime editing are imperative to develop new barley lines with superior qualities and better performance under stress conditions (Lin et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
The addition of N fertilizers has significantly increased crop production yields and is essential to maintain food security.
“The addition of N fertilizers has significantly increased crop production yields and is essential to maintain food security (He et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Introduction of CRISPR has greatly improved the functional characterization of genes.
“The introduction of CRISPR as a gene-editing platform has greatly improved the efficiency of breeding (Nonaka et al., 2017) and the functional characterization of genes (Zhang et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Only Vlčko and Ohnoutková (2020), Fu et al. (2022), and Karunarathne et al. (2022) had used CRISPR/Cas9 for barley germplasm development.
“The CRISPR/Cas9 system is a more precise technique than overexpression, yet only Vlčko and Ohnoutková (2020), Fu et al. (2022), and Karunarathne et al. (2022) had used this technology for barley germplasm development (Table 1).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al. (2022)
Researchers have sometimes used gene expression studies with reverse transcription-polymerase chain reaction (RT-PCR) or differential gene expression to determine potential genes for any given trait.
“Researchers have sometimes used gene expression studies with reverse transcription-polymerase chain reaction (RT-PCR) or differential gene expression to determine potential genes for any given trait (Feng et al., 2020a;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Downregulation of the ARE1 gene has been identified to improve NUE in cereals.
“For instance, downregulation of the ARE1 gene has been identified to improve NUE in cereals, which makes CRISPR/Cas9 gene knockout the best strategy to create loss of function mutant barley lines (Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
CRISPR/Cas9 gene knockout is the best strategy to create loss of function mutant barley lines for ARE1 downregulation.
“For instance, downregulation of the ARE1 gene has been identified to improve NUE in cereals, which makes CRISPR/Cas9 gene knockout the best strategy to create loss of function mutant barley lines (Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Other cultivars such as 'Compass', 'Spartacus', and the breeding line WI4330 have been used in previous research, but with low efficiency.
“Other cultivars such as 'Compass', 'Spartacus', and the breeding line WI4330 have been used in previous research, but with low efficiency (Ismagul et al., 2014;Han et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Enzymes that can digest the wild-type DNA, but not the mutant DNA, can be used to pinpoint mutations as heterozygous or homozygous on agarose gels.
“Enzymes that can digest the wild-type DNA, but not the mutant DNA, can be used to pinpoint mutations as heterozygous or homozygous on agarose gels (Han et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Several molecular techniques from agarose gel electrophoresis to sequencing can be used for genotyping mutants.
“Several molecular techniques from agarose gel electrophoresis to sequencing can be used for this task (Zhang et al., 2021;Karunarathne et al., 2022).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Karunarathne et al., 2022
Cites
MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms
Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018) · Molecular Biology and Evolution · doi:10.1093/molbev/msy096 · Cited by 37,683 · see citing works
Rapid isolation of high molecular weight plant DNA
Murray, M. G., & Thompson, W. F. (1980) · Nucleic Acids Research · doi:10.1093/nar/8.19.4321 · Cited by 12,156 · see citing works
Tamura, K., & Nei, M. (1993) · Molecular Biology and Evolution · doi:10.1093/oxfordjournals.molbev.a040023 · Cited by 11,267 · see citing works
Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133 · see citing works
Genetic screens in human cells using the CRISPR-Cas9 system
Wang, T., Wei, J. J., Sabatini, D. M., & Lander, E. S. (2014) · Science · doi:10.1126/science.1246981 · Cited by 2,856 · see citing works
Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014) · Nature Biotechnology · doi:10.1038/nbt.2969 · Cited by 2,072 · see citing works
Masclaux‐Daubresse, C., Daniel‐Vedele, F., Dechorgnat, J., Chardon, G., Gaufichon, L., & Suzuki, A. (2010) · Annals of Botany · doi:10.1093/aob/mcq028 · Cited by 1,773 · see citing works
A chromosome conformation capture ordered sequence of the barley genome
Mascher, M., Gundlach, H., Himmelbach, A., Beier, S., Twardziok, S. O., Wicker, T., Radchuk, V., Dockter, C., Hedley, P. E., Russell, J., et al. (2017) · Nature · doi:10.1038/nature22043 · Cited by 1,450 · see citing works
Self-citationA Modular Cloning System for Standardized Assembly of Multigene Constructs
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011) · PLoS ONE · doi:10.1371/journal.pone.0016765 · Cited by 1,385 · see citing works
Lane‐Serff, G. F., Rohling, E. J., Bryden, H. L., & Charnock, H. (1997) · Paleoceanography · doi:10.1029/96pa03934 · Cited by 1,049 · see citing works
Wang, Z.-P., Xing, H.-L., Li, D., Zhang, H.-Y., Han, C.-Y., Wang, X.-C., & Chen, Q.-J. (2015) · Genome biology · doi:10.1186/s13059-015-0715-0 · Cited by 1,036 · see citing works
Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes
Svitashev, S. K., Schwartz, C., Lenderts, B., Young, J. K., & Cigan, A. M. (2016) · Nature Communications · doi:10.1038/ncomms13274 · Cited by 826 · see citing works
Lawlor, D. W. (2002) · Journal of Experimental Botany · doi:10.1093/jexbot/53.370.773 · Cited by 766 · see citing works
Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice
Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H., & Yang, B. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku806 · Cited by 592 · see citing works
Lawrenson, T., Shorinola, O., Stacey, N., Li, C., Østergaard, L., Patron, N., Uauy, C., & Harwood, W. (2015) · Genome Biology · doi:10.1186/s13059-015-0826-7 · Cited by 551 · see citing works
Self-citationEditing plant genomes with CRISPR/Cas9
Belhaj, K., Chaparro‐Garcia, A., Kamoun, S., Patron, N. J., & Nekrasov, V. (2014) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2014.11.007 · Cited by 541 · see citing works
The regulation of nitrate and ammonium transport systems in plants
Glass, A. D. M. (2002) · Journal of Experimental Botany · doi:10.1093/jexbot/53.370.855 · Cited by 499 · see citing works
Pan, C., Ye, L., Li, Q., Liu, X., He, Y., Wang, J., Chen, L., & Lu, G. (2016) · Scientific Reports · doi:10.1038/srep24765 · Cited by 381 · see citing works
Wang, W., Hu, B., Yuan, D., Liu, Y., Che, R., Hu, Y., Ou, S., Liu, Y., Zhang, Z., Wang, H., et al. (2018) · The Plant Cell · doi:10.1105/tpc.17.00809 · Cited by 351 · see citing works
Travella, S., Ross, S., Harden, J., Everett, C. J., Snape, J. W., & Harwood, W. (2004) · Plant Cell Reports · doi:10.1007/s00299-004-0892-x · Cited by 255 · see citing works
He, X., Qu, B., Li, W., et al. (2015) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.00568 · Cited by 201 · see citing works
Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice
Wang, Q., Nian, J., Xie, X., Yu, H. J., Zhang, J., Bai, J., Dong, G., Hu, J., Bai, B., Chen, L., et al. (2018) · Nature Communications · doi:10.1038/s41467-017-02781-w · Cited by 147 · see citing works
Efficient generation of transgenic barley: The way forward to modulate plant–microbe interactions
Hensel, G., Valkov, V. T., Middlefell-Williams, J., & Kumlehn, J. (2007) · Journal of Plant Physiology · doi:10.1016/j.jplph.2007.06.015 · Cited by 140 · see citing works
Kapusi, E., Corcuera-Gómez, M., Melnik, S., & Stöger, E. (2017) · Frontiers in Plant Science · doi:10.3389/fpls.2017.00540 · Cited by 132 · see citing works
Zhang, J., Zhang, H., Li, S., Li, J., Yan, L., & Xia, L. (2021) · Journal of Integrative Plant Biology · doi:10.1111/jipb.13151 · Cited by 115 · see citing works
Han, Y., Yin, S., Huang, L., Wu, X., Zeng, J., Liu, X., Qiu, L., Munns, R., Chen, Z.-H., & Zhang, G. (2018) · Plant and Cell Physiology · doi:10.1093/pcp/pcy116 · Cited by 85 · see citing works
Self-citationMurray, F., Brettell, R. I. S., Matthews, P., Bishop, D., & Jacobsen, J. V. (2004) · Plant Cell Reports · doi:10.1007/s00299-003-0704-8 · Cited by 84 · see citing works
Shibghatallah, M. A. H., Khotimah, S. N., Suhandono, S., et al. (2013) · AIP conference proceedings · doi:10.1063/1.4820322 · Cited by 83 · see citing works
Wang, Q., Su, Q., Nian, J., Zhang, J., Guo, M., Dong, G., Hu, J., Wang, R., Wei, C., Li, G., et al. (2021) · Molecular Plant · doi:10.1016/j.molp.2021.04.012 · Cited by 83 · see citing works
Shinano, T., Kawamukai, T., Tadano, T., et al. (1996) · Photosynthetica · Cited by 78 · see citing works
A Protocol for High-Throughput Agrobacterium-Mediated Barley Transformation
Harwood, W. (2013) · Methods in molecular biology · doi:10.1007/978-1-62703-715-0_20 · Cited by 78 · see citing works
Yang, X., Xia, X., Zhang, Z., et al. (2017) · Frontiers in Plant Science · doi:10.3389/fpls.2017.01634 · Cited by 77 · see citing works
Chien, S. H., Teixeira, L. A. J., Cantarella, H., et al. (2016) · Agronomy Journal · doi:10.2134/agronj2015.0276 · Cited by 75 · see citing works
Highly efficient and genotype-independent barley gene editing based on anther culture
Han, Y., Broughton, S., Liu, L., Zhang, X.-Q., Zeng, J., He, X., & Li, C. (2021) · Plant Communications · doi:10.1016/j.xplc.2020.100082 · Cited by 73 · see citing works
Self-citationNitrogen economy in old and modern malting barleys
Abeledo, L. G., Calderini, D. F., & Slafer, G. A. (2008) · Field Crops Research · doi:10.1016/j.fcr.2007.11.006 · Cited by 72 · see citing works
A missense mutation in Large Grain Size 1 increases grain size and enhances cold tolerance in rice
Chen, X., Jiang, L., Zheng, J., et al. (2019) · Journal of Experimental Botany · doi:10.1093/jxb/erz192 · Cited by 70 · see citing works
Grashoff, C., & D’Antuono, L. F. (1997) · European Journal of Agronomy · doi:10.1016/s1161-0301(97)00001-4 · Cited by 66 · see citing works
Review: Strategies to increase nitrogen use efficiency of spring barley
Anbessa, Y., & Juskiw, P. E. (2012) · Canadian Journal of Plant Science · doi:10.4141/cjps2011-207 · Cited by 58 · see citing works
Zhang, S., Zhang, R., Gao, J., et al. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20174257 · Cited by 58 · see citing works
Wu, M., Liu, H., Lin, Y., et al. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.00307 · Cited by 42 · see citing works
Karunarathne, S. D., Han, Y., Zhang, X.-Q., Zhou, G., Hill, C. B., Chen, K., Angessa, T., & Li, C. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.571912 · Cited by 34 · see citing works
Self-citationDifferent knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes
Jiang, M., Hu, H., Jing, K., Traw, M. B., Yang, S., & Zhang, X. (2019) · Plant Molecular Biology · doi:10.1007/s11103-019-00871-5 · Cited by 33 · see citing works
Self-citationHan, Y. N., Jin, X., Wu, F., et al. (2011) · Journal of Zhejiang University SCIENCE B · doi:10.1631/jzus.b1000219 · Cited by 30 · see citing works
Self-citationExtraction and estimation of chlorophyll content of seed treated lentil crop using DMSO and acetone
Kumari, R., Ashraf, S., Bagri, G., et al. (2018) · Journal of Pharmacognosy and Phytochemistry · Cited by 28 · see citing works
Karunarathne, S. D., Han, Y. N., Zhang, X., & Li, C. (2020) · Agronomy · doi:10.3390/agronomy10050662 · Cited by 27 · see citing works
Self-citationHarada, K., Arizono, T., Sato, R., et al. (2019) · Plant and Cell Physiology · doi:10.1093/pcp/pcz203 · Cited by 23 · see citing works
Genetic manipulations of TaARE1 boost nitrogen utilization and grain yield in wheat
Guo, M., Wang, Q., Zong, Y., Nian, J., Li, H., Li, J., Wang, T., Gao, C., & Zuo, J. (2021) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2021.07.003 · Cited by 21 · see citing works
Rostami, H., Giri, A., Nejad, A. S. M., et al. (2013) · Saudi Journal of Biological Sciences · doi:10.1016/j.sjbs.2013.02.008 · Cited by 20 · see citing works
TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley
Orman-Ligeza, B., Harwood, W., Hedley, P. E., Hinchliffe, A., MacAulay, M. A., Uauy, C., & Trafford, K. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.00355 · Cited by 20 · see citing works
Genetic Variation in Grain Yield and Quality Traits of Spring Malting Barley
Hu, Y., Barmeier, G., & Schmidhalter, U. (2021) · Agronomy · doi:10.3390/agronomy11061177 · Cited by 19 · see citing works
BarleyVarDB: a database of barley genomic variation
Tan, C., Chapman, B., Wang, P., Zhang, Q., Zhou, G., Zhang, X., Barrero, R. A., Bellgard, M., & Li, C. (2020) · Database · doi:10.1093/database/baaa091 · Cited by 16 · see citing works
Self-citationWatt, C., Zhou, G., Angessa, T. T., Moody, D. B., & Li, C. (2020) · Australian Journal of Agricultural Research · doi:10.1071/cp20169 · Cited by 14 · see citing works
Self-citationAgrobacterium-Mediated Transformation of Barley (Hordeum vulgare L.)
Ismagul, A., Mazonka, I., Callegari, C., & Eliby, S. (2014) · Methods in Molecular Biology · doi:10.1007/978-1-4939-0446-4_16 · Cited by 11 · see citing works
Callus Induction and Plant Regeneration from Barley Mature Embryos (Hordem VulgareL.)
Abumhadi, N., Kamenarova, K., Todorovska, E., et al. (2005) · Biotechnology & Biotechnological Equipment · doi:10.1080/13102818.2005.10817224 · Cited by 9 · see citing works
Kiran, T. V., Vijayalakshmi, P., Rao, Y., et al. (2016) · Asian Research Journal of Agriculture · doi:10.9734/arja/2016/28503 · Cited by 5 · see citing works
Genetic analysis of tissue culture responses in barley (Hordeum vulgare L.).
Al-Nahdi, M. S. (2006) · Adelaide Research & Scholarship (AR&S) (University of Adelaide) · Cited by 1 · see citing works
Development of Cas9-expressing plants to study gene function
Chakraborty, A. (2020) · Murdoch University Research Portal · Cited by 1 · see citing works
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Karunarathne, S., Walker, E., Sharma, D., Li, C., & Han, Y. (2023) · Journal of Zhejiang University-SCIENCE B · doi:10.1631/jzus.b2200552 · Cited by 18 · see citing works
“The introduction of CRISPR as a gene-editing platform has greatly improved the efficiency of breeding (Non‐ aka et al., 2017) and the functional characterization of genes (Zhang et al., 2021; Karunarathne et al., 2022).”
InfluentialSelf-citation
- Authors
- Karunarathne, S. D., Han, Y., Zhang, X., & Li, C.
- Affiliations
- Murdoch University (AU)
- Western Crop Genetics Alliance, College of Science, Health, Engineering and Education Murdoch University Perth WA 6150 Australia
- Corresponding author
- Li, Chengdao
- Venue
- Journal of Integrative Plant Biology
- Publisher
- Wiley
- Published
- 2022-02-08
- Volume
- 64
- Issue
- 3
- Pages
- 756-770
- ISSN
- 0577-7496 (ISSN-L)
- 1672-9072
- 1744-7909
- OpenAlex
- W4205205304
- Cited by
- 61
- References
- 57
- Citations by year
- 2026: 16 · 2025: 12 · 2024: 13 · 2023: 9 · 2022: 11
- FWCI
- 6.20
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Integrative Plant Biology (publishedVersion)
- PubMed (publishedVersion)
- Murdoch University Research Portal (submittedVersion)
- Topics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant nutrient uptake and metabolism
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Life Sciences › Agricultural and Biological Sciences › Agronomy and Crop Science › Crop Yield and Soil Fertility
- Keywords
- barley, nitrogen use efficiency, cytokinin signaling, natural variation, CRISPR/Cas9 genome editing, leaf senescence, chlorophyll content, grain protein content, plant height, tillering, low nitrogen, nitrogen fertilizer reduction
- MeSH terms
- Gene Editing, Hordeum, Nitrogen, Genome-Wide Association Study, CRISPR-Cas Systems
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