Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis
Leaf rolling is a crucial agronomic trait to consider in rice (<i>Oryza sativa</i> L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The <i>SEMI-ROLLED LEAF 1</i> (<i>SRL1</i>) gene plays a key role in...
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/13/11087 |
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| author | Yue Han Jinlian Yang Hu Wu Fang Liu Baoxiang Qin Rongbai Li |
| author_facet | Yue Han Jinlian Yang Hu Wu Fang Liu Baoxiang Qin Rongbai Li |
| author_sort | Yue Han |
| collection | DOAJ |
| description | Leaf rolling is a crucial agronomic trait to consider in rice (<i>Oryza sativa</i> L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The <i>SEMI-ROLLED LEAF 1</i> (<i>SRL1</i>) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the <i>SRL1</i> gene in the indica rice line GXU103, which resulted in the generation of 14 T<sub>0</sub> transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T<sub>1</sub> generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting <i>SRL1</i> homozygous double-target knockout as <i>srl1-103</i>. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T<sub>2</sub> generation of <i>srl1-103</i> varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of <i>SRL1</i> in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T<sub>2</sub> generation of <i>srl1-103</i>. A comparison of <i>srl1-103</i> with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (<i>LOC_Os08g01670</i>, <i>LOC_Os05g46510</i>, <i>LOC_Os04g51450</i>, <i>LOC_Os10g28080</i>, <i>LOC_Os04g39814</i>, <i>LOC_Os01g71474</i>, <i>LOC_Os01g71350</i>, and <i>LOC_Os11g47600</i>) and vacuole-related genes (<i>LOC_Os09g23300</i>), which may partially explain the increased leaf rolling in <i>srl1-103</i>. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (<i>LOC_Os08g44840</i>) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which <i>SRL1</i> participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement. |
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| spelling | doaj.art-0faf3c49d2b8449fb8244ef7d391a5b62023-11-18T16:47:56ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124131108710.3390/ijms241311087Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome AnalysisYue Han0Jinlian Yang1Hu Wu2Fang Liu3Baoxiang Qin4Rongbai Li5State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, ChinaLeaf rolling is a crucial agronomic trait to consider in rice (<i>Oryza sativa</i> L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The <i>SEMI-ROLLED LEAF 1</i> (<i>SRL1</i>) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the <i>SRL1</i> gene in the indica rice line GXU103, which resulted in the generation of 14 T<sub>0</sub> transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T<sub>1</sub> generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting <i>SRL1</i> homozygous double-target knockout as <i>srl1-103</i>. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T<sub>2</sub> generation of <i>srl1-103</i> varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of <i>SRL1</i> in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T<sub>2</sub> generation of <i>srl1-103</i>. A comparison of <i>srl1-103</i> with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (<i>LOC_Os08g01670</i>, <i>LOC_Os05g46510</i>, <i>LOC_Os04g51450</i>, <i>LOC_Os10g28080</i>, <i>LOC_Os04g39814</i>, <i>LOC_Os01g71474</i>, <i>LOC_Os01g71350</i>, and <i>LOC_Os11g47600</i>) and vacuole-related genes (<i>LOC_Os09g23300</i>), which may partially explain the increased leaf rolling in <i>srl1-103</i>. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (<i>LOC_Os08g44840</i>) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which <i>SRL1</i> participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.https://www.mdpi.com/1422-0067/24/13/11087riceCRISPR/Cas9<i>SRL1</i>transcriptomeleaf rolling |
| spellingShingle | Yue Han Jinlian Yang Hu Wu Fang Liu Baoxiang Qin Rongbai Li Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis International Journal of Molecular Sciences rice CRISPR/Cas9 <i>SRL1</i> transcriptome leaf rolling |
| title | Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis |
| title_full | Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis |
| title_fullStr | Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis |
| title_full_unstemmed | Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis |
| title_short | Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of <i>SRL1</i> and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis |
| title_sort | improving rice leaf shape using crispr cas9 mediated genome editing of i srl1 i and characterizing its regulatory network involved in leaf rolling through transcriptome analysis |
| topic | rice CRISPR/Cas9 <i>SRL1</i> transcriptome leaf rolling |
| url | https://www.mdpi.com/1422-0067/24/13/11087 |
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