Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.

Leaf rolling is a crucial agronomic trait to consider in rice ( L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The () 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 gene in the indica rice line GXU103, which resulted in the generation of 14 T transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting homozygous double-target knockout as . 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 generation of 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 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T generation of . A comparison of 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 (, , , , , , , and ) and vacuole-related genes (), which may partially explain the increased leaf rolling in . Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene () 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 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.