CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean

ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.