Loss of leaf inclination 2 function leads to abaxial leaf rolling in rice due to upregulation of OsZHD1 and association with phenylpropanoid and flavonoid metabolism

Leaf rolling is a crucial agronomic trait influencing rice yield; moderate leaf rolling facilitates drought resistance and the formation of more erect plant architecture. This study utilized CRISPR/Cas9 gene editing to induce frameshift mutations in the leaf inclination 2 ( LC2 ) of rice ( Oryza sativa L.) cultivar Nipponbare. Cytological analysis revealed that lc2 mutants exhibited abaxial leaf rolling (outward rolling) and a significantly increased leaf rolling index (LRI) due to a greater number and larger size of bulliform cells compared to the wild-type (WT). Transcriptomic analysis showed that the loss of LC2 function remarkably upregulated the expression of OsZHD1 , whose overexpression directly induces abaxial leaf rolling in rice; Furthermore, we demonstrate that LC2 negatively regulate the expression of OsZHD1 via H3K27me3 modification of whole OsZHD1 gene and by occupying putative strong enhancers within the 3’ untranslated region (UTR) of OsZHD1 gene. Additionally, the expressions of two auxin efflux carrier genes, OsPIN1b and OsPILS7a , were also considerably upregulated in the lc2 mutant. Integrated transcriptomic and metabolomic analyses demonstrated that the expression levels of core structural genes in phenylpropanoid (e.g., OsPAL , OsPTAL ) metabolism and the flavonoid biosynthetic branch (e.g., OsCHS , OsCHI , OsF3’H , OsF3’H9 , and CYP98A ) were significantly upregulated in flag leaves of the lc2 mutant at the booting stage compared to the WT. This upregulation is associated with a significant accumulation of caffeoylquinic acid, commonly referred to as chlorogenic acid, which serves as a crucial precursor for lignin synthesis, thereby involves in the development of bulliform cells and associates with the abaxial leaf rolling observed in the lc2 mutant. Our current study addresses critical knowledge gaps concerning the multifunctionality of LC2 , and provides novel insights into the morphogenesis of abaxial leaf rolling.