CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)
Abstract
:1. Introduction
2. Material and Methods
2.1. Plant Material and Growth Conditions
2.2. CRISPR/Cas9 Plasmid Construction and Generation of Mutant Plants
2.3. Genotyping of T0 Generation and Off-target Effect Detection
2.4. Morphophysiological and Microscopic Analysis
2.5. Measurement of ABA Contents, MDA Contents, and Antioxidant Enzyme Assays Under Drought Stress
2.6. Protein Extraction, Digestion, and Labeling
2.7. Protein–Protein Interaction (PPI) and Data Analysis
2.8. Expression Analysis of Target Genes and Validation of Proteomic Data
2.9. Development of Hybrids and Assessment of Main Agronomic Characters
3. Results
3.1. Assembly of Targets in pYLCRISPR/Cas9Pubi-H
3.2. Frequency of Mutations and Off-target Analysis
3.3. Screening of T-DNA-Free Plants
3.4. Phenotyping Under Normal and Drought Conditions
3.5. Measurement of Chlorophyll Contents
3.6. Microscopic Analysis
3.7. Mutant Plants Enhance ABA Content and Improve Antioxidant Enzyme Activities Under Drought Stress
3.8. Protein Identification and Quantitation of Rolled Leaf Mutant and Wild Type
3.9. Functional Networks of the Differentially Expressed Proteins
3.10. Gene Ontology (GO) Annotation of Up- and Downregulated Proteins
3.11. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis of Up- and Downregulated Proteins
3.12. Hub-Protein Analysis
3.13. Expression Analysis of Target Genes Quantitative Real-Time-PCR Validation of DEPs
3.14. Performance of Semi-Rolled Leaf Hybrids Produced by Wild Type and Mutant Rolled Leaf Restorers
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Accession Numbers
References
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Genotypes | LT | PH | PN | PL (cm) | FLL (cm) | FLW (cm) | GNPP | SSR (%) | GW (g) | GL (mm) | GWD (mm) | GYPP (g) |
---|---|---|---|---|---|---|---|---|---|---|---|---|
GXU16 | Flat | 112.1 ± 3.4 | 7.5 ± 1.3 | 25.6 ± 1.4 | 51.0 ± 3.6 | 2.2 ± 0.1 * | 137 ± 12 | 90.2 ± 8.9 | 32.5 ± 1.3 | 10.0 ± 0.1 | 3.3 ± 0.2 | 30.0 ± 0.5 |
GXU20 | Flat | 118.1 ± 2.6 | 7.8 ± 1.7 | 28.3 ± 1.5 | 48.5 ± 2.6 | 2.3 ± 0.3 | 139 ± 15 | 91.4 ± 5.3 | 34.5 ± 1.5 | 11.0 ± 0.3 | 3.4 ± 0.1 | 33.9 ± 0.1 |
GXU28 | Flat | 125.2 ± 3.4 | 7.7 ± 2.0 | 27.4 ± 1.2 | 48.3 ± 1.9 | 2.4 ± 0.6 | 138 ± 10 | 90.5 ± 5.6 | 33.4 ± 1.4 | 11.0 ± 0.2 | 3.4 ± 0.3 | 32.8 ± 0.6 |
GXU16-19 | Rolled | 80.1 ± 5.4 * | 10.8 ± 1.0 * | 17.4 ± 1.2 * | 37.5 ± 1.3 * | 1.6 ± 0.3 * | 115 ± 10 * | 72.3 ± 6.7 * | 28.5 ± 1.2 * | 9.1 ± 0.6 * | 3.0 ± 0.5 * | 25.4 ± 0.1 * |
GXU20-8 | Rolled | 83.2 ± 4.3 * | 11.3 ± 1.0 * | 16.2 ± 1.2 * | 37.3 ± 1.8 * | 1.7 ± 0.5 * | 116 ± 11 * | 69.5 ± 5.3 * | 29.6 ± 1.5 * | 9.2 ± 0.8 * | 2.9 ± 0.4 * | 27.3 ± 0.1 * |
GXU28-12 | Rolled | 87.3 ± 3.9 * | 10.4 ± 2.0 * | 17.5 ± 1.4 * | 36.9 ± 1.7 * | 1.7 ± 0.6 * | 109 ± 13 * | 67.6 ± 3.6 * | 28.7 ± 1.3 * | 9.2 ± 0.5 * | 2.9 ± 0.8 * | 21.9 ± 0.4 * |
Genotypes | GXU16 | GXU20 | GXU28 | GXU16-19 | GXU20-8 | GXU28-12 |
---|---|---|---|---|---|---|
Chl a (mg·g−1) | 8.26 ± 0.11 | 8.13 ± 0.08 | 7.99 ± 0.07 | 4.25 ± 0.33 * | 4.11 ± 0.16 * | 4.14 ± 0.28 * |
Chl b (mg·g−1) | 2.29 ± 0.03 | 2.75 ± 0.05 | 2.86 ± 0.12 | 1.52 ± 0.03 * | 1.49 ± 0.11 * | 1.39 ± 0.05 * |
β-Car (mg·g−1) | 1.39 ± 0.06 | 1.47 ± 0.05 | 1.36 ± 0.04 | 0.98 ± 0.05 * | 1.02 ± 0.04 * | 1.06 ± 0.02 * |
Chl a+Chl b (mg·g−1) | 10.54 ± 0.12 | 10.87± 0.09 | 10.85 ± 0.11 | 5.77 ± 0.07 * | 5.60 ± 0.08 * | 5.53 ± 0.03 * |
Chl a/Chl b | 3.60 ± 0.12 | 2.95 ± 0.09 | 2.79 ± 0.06 | 2.79 ± 0.03 * | 2.75 ± 0.07 * | 2.97 ± 0.08 * |
Protein ID | Gene Name/Locus | Annotation | Regulation |
---|---|---|---|
Proteins involved in abiotic stress | |||
B8APG3 | OsI_11487 | Peroxidase | Up |
B8ARU3 | OsI_18017 | Peroxidase | Up |
A2YY59 | OsI_30285 | Superoxide dismutase | Up |
A2ZBV1 | ASR5 | Abscisic stress-ripening protein 5 | Up |
B8BFT1 | OsI_32804 | HSP domain-containing protein | Up |
A2X294 | OsI_06318 | Hydrolase_4 domain-containing protein | Up |
B8B3F7 | OsI_21871 | ParB domain-containing protein | Up |
B8BBZ9 | OsI_28223 | NAD(P)-binding domain-containing protein | Up |
Proteins involved in leaf rolling and development | |||
Q8L3S3 | Os01g0572800 | Lateral organ boundaries (LOB) domain protein-like | Down |
Q7XGL4 | CRLL2 | LOB domain protein 31 | Down |
Q852M3 | LOC_Os03g57670 | LOB domain protein 4 | Down |
Q8L4M5 | B1129G05.33 | LOB domain-containing protein | Down |
A2X266 | OsI_06294 | Glycosyltransferase | Down |
A2WLD5 | OsI_00644 | Glycosyltransferase | Down |
B8BCU4 | OsI_31813 | Glycosyltransferase | Down |
A2ZFX4 | OsI_36677 | Glycosyltransferase | Up |
A2Y650 | OsI_20474 | Phosphoglycerate kinase | Down |
A2Z498 | OsI_32475 | Peptidyl-prolyl cis-trans isomerase | Down |
A2X9U8 | OsI_09019 | Peptidylprolyl isomerase | Down |
A2XZE6 | OsI_18093 | Peptidyl-prolyl cis-trans isomerase | Down |
A2YCR4 | OsI_22900 | Acyl-coenzyme A oxidase | Down |
Genotypes | LT | PH | PN | PL (cm) | GNPP | SSR (%) | GW (g) | GL (mm) | GWD (mm) | GYPP (g) |
---|---|---|---|---|---|---|---|---|---|---|
GXU16 | Flat | 117.7 ± 4.2 | 7.6 ± 1.2 | 25.6 ± 1.4 | 134 ± 10 | 88.2 ± 8.7 | 32.5 ± 1.2 | 10.0 ± 0.1 | 3.1 ± 0.2 | 31.0 ± 0.3 |
GXU20 | Flat | 119.3 ± 3.6 | 7.5 ± 1.6 | 28.3 ± 1.5 | 132 ± 12 | 89.4 ± 5.1 | 31.5 ± 1.6 | 11.0 ± 0.3 | 2.9 ± 0.1 | 33.9 ± 0.2 |
GXU28 | Flat | 124.2 ± 3.7 | 7.7 ± 1.3 | 27.4 ± 1.2 | 133 ± 11 | 90.5 ± 5.2 | 31.4 ± 1.2 | 11.0 ± 0.2 | 3.0 ± 0.2 | 33.8 ± 0.5 |
36A/GXU16 | Flat | 114.6 ± 3.8 NS | 7.8 ± 1.6 NS | 26.1 ± 1.1 NS | 140 ± 18 NS | 83.3 ± 2.8 NS | 32.8 ± 1.0 NS | 10.0 ± 0.1 NS | 2.8 ± 0.1 NS | 29.8 ± 0.4 NS |
52A/GXU16 | Flat | 116.0 ± 3.6 NS | 8.0 ± 1.4 NS | 25.5 ± 1.3 NS | 145 ± 25 NS | 86.4 ± 2.6 NS | 33.2 ± 1.1 NS | 10.2 ± 0.2 NS | 2.8 ± 0.2 NS | 33.3 ± 0.4 NS |
36A/GXU20 | Flat | 115.2 ± 2.7 NS | 7.9 ± 2.1 NS | 26.3 ± 1.0 NS | 149 ± 21 NS | 83.8 ± 3.8 NS | 32.4 ± 1.2 NS | 10.0 ± 0.2 NS | 2.9 ± 0.2 NS | 31.9 ± 0.5 NS |
52A/GXU20 | Flat | 121.4 ± 5.0 NS | 8.8 ± 1.2 NS | 24.8 ± 1.6 NS | 134 ± 22 NS | 82.7 ± 4.4 NS | 32.5 ± 1.2 NS | 10.1 ± 0.3 NS | 2.8 ± 0.1 NS | 31.7 ± 0.4 NS |
36A/GXU28 | Flat | 115.2 ± 4.4 NS | 8.0 ± 0.9 NS | 25.5 ± 1.2 NS | 146 ± 21 NS | 85.4 ± 3.3 NS | 29.8 ± 1.3 NS | 10.0 ± 0.3 NS | 2.9 ± 0.2 NS | 29.7 ± 0.4 NS |
52A/GXU28 | Flat | 122.4 ± 5.9 NS | 8.2 ± 1.0 NS | 25.2 ± 1.6 NS | 138 ± 27 NS | 86.0 ± 3.2 NS | 29.4 ± 1.3 NS | 10.2 ± 0.3 NS | 2.9 ± 0.2 NS | 28.6 ± 0.6 NS |
36A/GXU16-19 | SR | 110.1 ± 5.5 NS | 8.8 ± 1.8 * | 28.3 ± 1.2 NS | 166 ± 12 * | 84.0 ± 3.8 NS | 31.6 ± 1.0 NS | 10.0 ± 0.1NS | 2.9 ± 0.1 NS | 38.7 ± 0.4 * |
52A/GXU16-19 | SR | 122.1 ± 3.6 NS | 10.2 ± 1.9 * | 27.6 ± 1.0 NS | 158 ± 12 * | 85.2 ± 4.2 NS | 32.4 ± 1.2 NS | 9.8 ± 0.2 NS | 2.8 ± 0.1 NS | 44.5 ± 0.4 * |
36A/GXU20-8 | SR | 112.5 ± 5.5 NS | 9.2 ± 1.8 * | 28.5 ± 1.4 NS | 168 ± 14 * | 85.4 ± 3.3 NS | 32.5 ± 1.0 NS | 10.1 ± 0.2 NS | 2.9 ± 0.1 NS | 42.9 ± 0.4 * |
52A/GXU20-8 | SR | 126.1 ± 5.0 NS | 10.5 ± 1.3 * | 27.0 ± 1.1 NS | 156 ± 18 * | 84.5 ± 4.9 NS | 32.6 ± 1.3 NS | 10.0 ± 0.1 NS | 2.8 ± 0.2 NS | 43.4 ± 0.4 * |
36A/GXU28-12 | SR | 117.8 ± 4.8 NS | 9.0 ± 1.1 * | 28.8 ± 1.0 NS | 170 ± 15 * | 86.7 ± 3.4 NS | 32.3 ± 1.1 NS | 9.9 ± 0.2 NS | 2.9 ± 0.2 NS | 40.6 ± 0.4 * |
52A/GXU28-12 | SR | 126.6 ± 3.4 NS | 10.0 ± 1.2 * | 27.2 ± 0.9 NS | 153 ± 14 * | 86.9 ± 6.0 NS | 31.5 ± 1.2 NS | 10.1 ± 0.2 NS | 2.8 ± 0.1 NS | 40.6 ± 0.4 * |
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Liao, S.; Qin, X.; Luo, L.; Han, Y.; Wang, X.; Usman, B.; Nawaz, G.; Zhao, N.; Liu, Y.; Li, R. CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.). Agronomy 2019, 9, 728. https://doi.org/10.3390/agronomy9110728
Liao S, Qin X, Luo L, Han Y, Wang X, Usman B, Nawaz G, Zhao N, Liu Y, Li R. CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.). Agronomy. 2019; 9(11):728. https://doi.org/10.3390/agronomy9110728
Chicago/Turabian StyleLiao, Shanyue, Xuemei Qin, Liang Luo, Yue Han, Xin Wang, Babar Usman, Gul Nawaz, Neng Zhao, Yaoguang Liu, and Rongbai Li. 2019. "CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)" Agronomy 9, no. 11: 728. https://doi.org/10.3390/agronomy9110728