Transcriptomic and Phenotypic Analysis of Maize with CRISPR/Cas9-mediated Targeted Mutagenesis of Melatonin Synthesis Genes under Drought Stress

Melatonin, a pleiotropic regulatory factor, plays a key role in mediating crop drought resistance. Herein, we conducted an integrated physiological and transcriptomic approach to elucidate the mitigating effect of endogenous melatonin in mitigating drought stress in maize. We generated a comt snat asmt maize mutant via CRISPR-Cas9-mediated simultaneous editing of ZmCOMT , ZmSNAT , and ZmASMT1 -genes encoding rate-limiting enzymes in the endogenous melatonin biosynthesis pathway. Sequencing of the mutant lines revealed key amino acid substitutions (Gly168→Ala in ZmSNAT , Asp175→Glu in ZmCOMT , and Asp150→Glu in ZmASMT1 ) within critical protein domains, resulting from CRISPR-induced small insertions or deletions (indels), which led to subtle alterations in the tertiary conformation of corresponding proteins. These modifications resulted in an 86.70% increase in endogenous melatonin content. Under drought stress, the comt snat asmt maize exhibited enhanced antioxidant enzyme activities, leading to a significant reduction in reactive oxygen species (ROS) accumulation compared to the control. Furthermore, endogenous levels of melatonin, abscisic acid (ABA), cytokinin (CTK), and auxin (IAA) were markedly elevated, whereas gibberellin (GA) content was significantly reduced. Consistently, the activities of SNAT, ASMT, and COMT were also enhanced in the mutant. Transcriptomic profiling further revealed that endogenous melatonin regulates ABA, IAA, CTK, and GA signaling pathways to enhance drought tolerance. In particular, ZmCOMT , ZmSNAT , and ZmASMT1 apparently modulated the expression levels of key regulatory genes such as ZmIAA2 , ZmIAA23 , ZmIAA7 , ZmSAUR24 , ZmPYL8 , and ZmPIF3.1 , associated with these hormone pathways . Collectively, endogenous melatonin reinforces drought tolerance by reducing ROS accumulation and reprogramming phytohormone homeostasis through regulation of hormone-related gene expression. Our findings provide important insights into the regulatory mechanisms by which endogenous melatonin enhances drought resistance in crops. • Simultaneous CRISPR/Cas9 editing of three key genes ( ZmCOMT , ZmSNAT , and ZmASMT1 ) generated gain-of-function mutants with enhanced melatonin biosynthesis and drought tolerance.; • The 86.7% increase in endogenous melatonin orchestrated drought resilience by boosting the antioxidant system and reprogramming phytohormone signaling networks; • Integrated transcriptomic and physiological analyses revealed that melatonin-mediated hormone signaling fine-tuned growth-defense balance under drought stress.