Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis

Abstract

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) 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 SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T₀ transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T₁ generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. 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 T₂ generation of srl1-103 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 SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T₂ generation of srl1-103. A comparison of srl1-103 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 (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) 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 SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

Keywords: CRISPR/Cas9; SRL1; leaf rolling; rice; transcriptome.

Figure 1

sgRNA design for SEMI-ROLLED LEAF 1 (SRL1) target sequences and CRISPR/Cas9 vector construction. (A) The gene structure of SRL1, as well as the sequence and location of the targets. (B) The sgRNA expression cassettes for the two targets, U6a (629 bp) and U6b (515 bp). (C) Construction of CRISPR/Cas9 knockout vector.

Figure 2

Target mutation types of T₀ double-target mutant plants, and identification and screening of T₀ and T₁ generations. (A) Sequence schematic of the targets in SRL1 and the types of double-target mutations. Targets are in red boxes, PAMs are underlined in blue, insertion and deletion nucleotides are in red boxes. (B) Amplification results of partial T₀ generation plants using specific primers hyg-F/hyg-R (417 bp PCR product). (C) Exogenous screening of partial T₁ generation using specific primers Cas9-F/Cas9-R (572 bp PCR product). Marker: 2000 bp. The numbers 1–11 represent the lanes. Wild type: WT.

Figure 3

Physiological morphology and agronomic traits of the wild-type GXU103 and the mutant line srl1-103. (A) Plant at maturity, bar = 10 cm. (B) Frontal photograph of mature flag leaf, bar = 2 cm. (C) Section of mature flag leaf, bar = 2 mm. (D) Cross-section of leaf blade, bar = 100 μm. Bulliform cells between vascular bundles are in red boxes. (E–L) Agronomic traits of the wild-type GXU103 and the mutant line srl1-103: plant height, length of flag leaf, width of flag leaf, leaf-rolling index, number of panicles, length of panicle, seed setting rate, 1000-grain weight. Statistical analysis used Student’s t test to determine significant differences (* p < 0.05, ** p < 0.01).

Figure 4

Overview of RNA-seq data analysis. (A) Pair-wise correlation analysis (Pearson’s correlation coefficient) of all the samples. (B) Genes that showed significant differential expression between GXU103 and srl1-103. Red dots indicate upregulated genes. Blue dots indicate downregulated genes. Gray dots indicate genes with no significant differences. (C) Gene ontology enrichment analysis of GXU103 and srl1-103.

Figure 5

Predicted gene regulatory networks involving SRL1. PG: polygalacturonase. GH: glycosyl hydrolase.