CoChat
Sign Up
Cited by 1,029CRISPR and Genetic EngineeringPlant Stress Responses and ToleranceGenetically Modified Organisms Research

The DOI matches its Crossref record; 56 of the 56 references it cites were found in a registry or an index.

ARGOS 8 variants generated by CRISPR ‐Cas9 improve maize grain yield under field drought stress conditions

Open access
Shi, Jinrui, Gao, Huirong, Wang, Hongyu, Lafitte, H. Renee, Archibald, Rayeann L., Yang, Meizhu, Hakimi, Salim M., Mo, Hua, Habben, Jeffrey E.·2017·Plant Biotechnology Journal
DOI 10.1111/pbi.12603PMC PMC5258859
Open this paper in CoChat — free accountOpen PDF

Abstract

Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas-enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought-tolerant crops.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1111/pbi.12603

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Maize ARGOS8 reduces plant responses to ethylene when overexpressed in transgenic Arabidopsis plants. (a) Ethylene triple response of Arabidopsis ARGOS8 transgenic plants ( ARGOS8) and wild‐type (WT) controls to 0.5 μm of the ethylene precursor aminocyclopropane‐1‐carboxylic acid (ACC). A short version of ARGOS8 was overexpressed under control of the cauliflower mosaic virus 35S promoter (35S). Composite image of representative 3‐day‐old etiolated seedlings. Bar = 2 mm. (b) Hypocotyl and root lengths of etiolated Arabidopsis seedlings overexpressing the short version of ARGOS8. Four transgenic lines (E1, E2, E4 and E12) and wild‐type (WT) controls were grown in the dark in the presence of indicated ACC concentrations for 3 days. Data are means ± SD, n = 15. Significant differences of the transgenic plants from the WT are denoted by asterisks (*P < 0.05, **P < 0.01, ANOVA, Tukey's HSD).
Figure 1 Maize ARGOS8 reduces plant responses to ethylene when overexpressed in transgenic Arabidopsis plants. (a) Ethylene triple response of Arabidopsis ARGOS8 transgenic plants ( ARGOS8) and wild‐type (WT) controls to 0.5 μm of the ethylene precursor aminocyclopropane‐1‐carboxylic acid (ACC). A short version of ARGOS8 was overexpressed under control of the cauliflower mosaic virus 35S promoter (35S). Composite image of representative 3‐day‐old etiolated seedlings. Bar = 2 mm. (b) Hypocotyl and root lengths of etiolated Arabidopsis seedlings overexpressing the short version of ARGOS8. Four transgenic lines (E1, E2, E4 and E12) and wild‐type (WT) controls were grown in the dark in the presence of indicated ACC concentrations for 3 days. Data are means ± SD, n = 15. Significant differences of the transgenic plants from the WT are denoted by asterisks (*P < 0.05, **P < 0.01, ANOVA, Tukey's HSD).CC BY · PubMed Central
Figure 2 Editing the ARGOS8 genomic sequence using the CRISPR/Cas9 system to generate variants with constitutive expression. (a) Schematic drawing illustrating the insertion of GOS2 PRO into the 5′‐UTR of ARGOS8 and the promoter swap. CTS, CRISPR‐RNA target site; HA, homology arm; HDR, homology‐directed repair; GOS2 PRO, maize GOS2 promoter and the 5′‐UTR with an intron. (b) Genomic sequence of the ARGOS8 5′‐UTR and the upstream region. The CRISPR‐RNA target sites (CTS) are highlighted in red, and the protospacer adjacent motifs (PAM) are shown in blue font. The ARGOS8 coding region is shown in bold font. (c) Diagram showing primers used in junction PCR for genotyping regenerated shoots and long PCR for amplifying and sequencing the entire modification region in homozygous plants. The relative position and direction of PCR primers (P) are indicated by arrows. P1 and P2 for the HR1 junction; P5 and P4 for the HR2 junction; P1 and P4 for the long PCR. (d) Junction PCR analysis of regenerated shoots. Agarose gel images are shown for representative regenerated shoots positive for one junction or two junctions and shoots negative in the junction PCR assay. JP1, HR1 junction PCR with the primer P1 and P2; JP2, HR2 junction PCR with P5 and P4. (e) PCR screening regenerated shoots for deletion in the ARGOS8 locus. An agarose gel image is shown for PCR products amplified with the primer P1 and P4 in representative shoots (Lanes 1‐14) generated from the CRISPR RNA‐3 and RNA‐1 transformation. M, DNA molecular weight markers.
Figure 2 Editing the ARGOS8 genomic sequence using the CRISPR/Cas9 system to generate variants with constitutive expression. (a) Schematic drawing illustrating the insertion of GOS2 PRO into the 5′‐UTR of ARGOS8 and the promoter swap. CTS, CRISPR‐RNA target site; HA, homology arm; HDR, homology‐directed repair; GOS2 PRO, maize GOS2 promoter and the 5′‐UTR with an intron. (b) Genomic sequence of the ARGOS8 5′‐UTR and the upstream region. The CRISPR‐RNA target sites (CTS) are highlighted in red, and the protospacer adjacent motifs (PAM) are shown in blue font. The ARGOS8 coding region is shown in bold font. (c) Diagram showing primers used in junction PCR for genotyping regenerated shoots and long PCR for amplifying and sequencing the entire modification region in homozygous plants. The relative position and direction of PCR primers (P) are indicated by arrows. P1 and P2 for the HR1 junction; P5 and P4 for the HR2 junction; P1 and P4 for the long PCR. (d) Junction PCR analysis of regenerated shoots. Agarose gel images are shown for representative regenerated shoots positive for one junction or two junctions and shoots negative in the junction PCR assay. JP1, HR1 junction PCR with the primer P1 and P2; JP2, HR2 junction PCR with P5 and P4. (e) PCR screening regenerated shoots for deletion in the ARGOS8 locus. An agarose gel image is shown for PCR products amplified with the primer P1 and P4 in representative shoots (Lanes 1‐14) generated from the CRISPR RNA‐3 and RNA‐1 transformation. M, DNA molecular weight markers.CC BY · PubMed Central
Figure 3 Maize genome‐edited ARGOS8 variants. (a) Genomic sequence upstream of the ARGOS8 coding region in three genome‐edited variants. The entire modification region in homozygous F2 plants was amplified using long PCR, and the PCR products were sequenced. Part of the GOS2 5′‐UTR sequence (blue font) and the remaining 5′‐UTR of ARGOS8 as well as the 5′‐terminus of ARGOS8 coding sequence are shown. In the promoter deletion variant ARGOS8‐v3, the remnant CTS3 and CTS1 sequences are highlighted. (b) Relative expression levels of ARGOS8 in leaves as measured by qRT‐PCR. Means ± SD are shown for F2 plants of 14‐day‐old ARGOS8‐v1 and 18‐day‐old ARGOS8‐v2; n = 10–24. WT, wild‐type; Hete, Heterozygote; Homo, homozygote.
Figure 3 Maize genome‐edited ARGOS8 variants. (a) Genomic sequence upstream of the ARGOS8 coding region in three genome‐edited variants. The entire modification region in homozygous F2 plants was amplified using long PCR, and the PCR products were sequenced. Part of the GOS2 5′‐UTR sequence (blue font) and the remaining 5′‐UTR of ARGOS8 as well as the 5′‐terminus of ARGOS8 coding sequence are shown. In the promoter deletion variant ARGOS8‐v3, the remnant CTS3 and CTS1 sequences are highlighted. (b) Relative expression levels of ARGOS8 in leaves as measured by qRT‐PCR. Means ± SD are shown for F2 plants of 14‐day‐old ARGOS8‐v1 and 18‐day‐old ARGOS8‐v2; n = 10–24. WT, wild‐type; Hete, Heterozygote; Homo, homozygote.CC BY · PubMed Central
Figure 4 Comparison of the ARGOS8 expression in genome‐edited variants and wild‐type maize plants. (a) Relative expression of ARGOS8 in a selection of maize tissues and stages. mRNA was quantified with qRT‐PCR. Six individual plants were analysed for the genome‐edited variants and two plants for WT controls. DAP, days after pollination. (b) ARGOS8 protein expression in developing kernels. Immature kernels (21 DAP) were analysed by immunoblotting using a monoclonal anti‐ARGOS8 antibody.
Figure 4 Comparison of the ARGOS8 expression in genome‐edited variants and wild‐type maize plants. (a) Relative expression of ARGOS8 in a selection of maize tissues and stages. mRNA was quantified with qRT‐PCR. Six individual plants were analysed for the genome‐edited variants and two plants for WT controls. DAP, days after pollination. (b) ARGOS8 protein expression in developing kernels. Immature kernels (21 DAP) were analysed by immunoblotting using a monoclonal anti‐ARGOS8 antibody.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 56 references

Cited by

Sorted by citations

Items 601–700 of 1,000

Related

See all

Metadata

Authors
Shi, J., Gao, H., Wang, H., Lafitte, H. R., Archibald, R. L., Yang, M., Hakimi, S. M., Mo, H., & Habben, J. E.
Affiliations
Pioneer Hi-Bred (US)
Corresponding author
Shi, Jinrui
ORCID
Venue
Plant Biotechnology Journal
Publisher
Wiley
Published
2016-08-17
Volume
15
Issue
2
Pages
207-216
ISSN
  • 1467-7644 (ISSN-L)
  • 1467-7652
DOI
10.1111/pbi.12603
PMC
PMC5258859
OpenAlex
W2488393959
Cited by
1,029
References
56
Citations by year
2026: 97 · 2025: 119 · 2024: 99 · 2023: 127 · 2022: 157 · 2021: 145 · 2020: 109 · 2019: 85 · 2018: 47 · 2017: 40 · 2016: 3
FWCI
30.33
Type
journal article
Language
en
Open access
Licence
Locations
  • Plant Biotechnology Journal (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Stress Responses and Tolerance
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
Keywords
maize, drought stress, grain yield, CRISPR/Cas9, ethylene response, promoter replacement, allelic variation, drought tolerance breeding, gene expression
MeSH terms
Gene Editing, Base Sequence, Edible Grain, Zea mays, Ethylenes, Plant Proteins, Promoter Regions, Genetic, RNA, Messenger, Stress, Physiological, Polymerase Chain Reaction, Gene Expression Regulation, Plant, Crops, Agricultural, Plants, Genetically Modified, Biodiversity, Droughts, CRISPR-Cas Systems, CRISPR-Associated Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review