Multiplexed CRISPR/Cas9-Mediated Knockout of Laccase Genes in Salvia miltiorrhiza Revealed Their Roles in Growth, Development, and Metabolism

Abstract

Laccases are multicopper-containing glycoproteins related to monolignol oxidation and polymerization. These properties indicate that laccases may be involved in the formation of important medicinal phenolic acid compounds in Salvia miltiorrhiza such as salvianolic acid B (SAB), which is used for cardiovascular disease treatment. To date, 29 laccases have been found in S. miltiorrhiza (SmLACs), and some of which (SmLAC7 and SmLAC20) have been reported to influence the synthesis of phenolic acids. Because of the functional redundancy of laccase genes, their roles in S. miltiorrhiza are poorly understood. In this study, the CRISPR/Cas9 system was used for targeting conserved domains to knockout multiple genes of laccase family in S. miltiorrhiza. The expressions of target laccase genes as well as the phenolic acid biosynthesis key genes decrease dramatically in editing lines. Additionally, the growth and development of hairy roots was significantly retarded in the gene-edited lines. The cross-sections examination of laccase mutant hairy roots showed that the root development was abnormal and the xylem cells in the edited lines became larger and looser than those in the wild type. Additionally, the accumulation of RA as well as SAB was decreased, and the lignin content was nearly undetectable. It suggested that SmLACs play key roles in development and lignin formation in the root of S. miltiorrhiza and they are necessary for phenolic acids biosynthesis.

Genome-wide association study in two-row spring barley landraces identifies QTL associated with plantlets root system architecture traits in well-watered and osmotic stress conditions

“…Laccases are enzymes involved in oxidative polymerization of lignin that accumulates particularly in cells where secondary thickening of cell wall occurs ( Zhao et al., 2013 ). The importance of laccase in root hairs and xylem development has been demonstrated in Salvia miltiorrhiza ( Zhou et al., 2021 ). Interestingly two tandem arrayed laccases were also located within the LD block 6H-488 that contains four high confidence genes.…”

Identification of Abietane-Type Diterpenoids and Phenolic Acids Biosynthesis Genes in Salvia apiana Jepson Through Full-Length Transcriptomic and Metabolomic Profiling

“…A BAHD acyltransferase member [rosmarinic acid synthase (RAS)] and CYP98A subfamily were reported to contribute to the specific PAs biosynthesis in Lamiaceae by producing rosmarinic acid. Inferring from the indirect evidences, laccase family might catalyze further polymerization (Di et al, 2013 ; Li et al, 2019 ; Zheng et al, 2021 ; Zhou et al, 2021 ); however, it still needs to be proved. The absence of salvianolic acid B in S. apiana implies the lose function of laccase family, which can be considered as a natural mutant.…”

CRISPR-Cas gene editing technology and its application prospect in medicinal plants

“…This result verified the function of SmRAS. In 2021, Zhou et al knocked out more than 20 genes of the laccase family in S. miltiorrhiza simultaneously by using the CRISPR-Cas9 dual-locus editing technology [121]. The expression levels of the target laccase genes and phenolic acid biosynthesis key genes and the accumulation of RA, SAB and lignin decreased dramatically in the editing lines.…”