Multi‐omics analysis identifies EcCS4 is negatively regulated in response to phytotoxin isovaleric acid stress in Echinochloa crus‐galli

Abstract BACKGROUND Knowledge of herbicidal targets is critical for weed management and food safety. The phytotoxin isovaleric acid (ISA) is effective against weeds with a broad spectrum, carries low environmental risks, and is thus an excellent herbicide lead. However, the biochemical and molecular mechanisms underlying the action of ISA remain unclear. RESULTS Multi‐omics data showed that acetyl coenzyme A (acetyl‐CoA) was the key affected metabolite, and that citrate synthase (CS) 4 was substantially down‐regulated under ISA treatment in Echinochloa crus‐galli leaves. In particular, the transcript level of EcCS4 was the most significantly regulated among the six genes involved in the top 10 different pathways. The EcCS4 encodes a protein of 472 amino acids and is localized to the cell membrane and mitochondria, similar to the CS4s of other plants. The protein content of EcCS4 was down‐regulated after ISA treatment at 0.5 h. ISA markedly inhibited the CS4 activity in vitro in a concentration‐dependent manner (IC 50 = 41.35 μM). In addition, the transgenic rice plants overexpressing EcCS4 (IC 50 = 111.8 mM for OECS4‐8 line) were more sensitive, whereas loss‐of‐function rice mutant lines (IC 50 = 746.5 mM for oscs4‐19 ) were more resistant to ISA, compared to wild type (WT) plants (IC 50 = 355.6 mM). CONCLUSION CS4 was first reported as a negative regulator of plant responses to ISA. These results highlight that CS4 is a candidate target gene for the development of novel herbicides and for breeding herbicide‐resistant crops. © 2023 Society of Chemical Industry.