Disruption of ACO2 and PI3K alters ABA-ethylene homeostasis and pre-harvest sprouting dynamics in rice

Pre-harvest sprouting (PHS) severely compromises rice grain quality when physiologically mature panicles are exposed to prolonged rainfall or high relative humidity.Although seed dormancy serves as a primary barrier against precocious germination, an extended initial delay in germination does not invariably confer sustained resistance to PHS.Here, we investigated the functions of 1-aminocyclopropane-1-carboxylate oxidase 2 (ACO2) and phosphatidylinositol 3-kinase (PI3K) in rice seed germination and PHS employing CRISPR/Cas9-mediated targeted mutagenesis.Transgene-free homozygous aco2#21-2 and pi3k#2-3 mutants were successfully generated in the japonica cultivar Dongjin.Relative to the wild type (WT), both mutants exhibited delayed germination, lower germination indices, higher dormancy indices, and increased sensitivity to exogenous abscisic acid (ABA).Ethylene production decreased sharply from 1.35 nL g -1 fresh weight (FW) h -1 in WT seeds to 0.16 and 0.45 nL g -1 FW h -1 in aco2#21-2 and pi3k#2-3, respectively, whereas ABA content increased from 38 to 123 and 82 ng g -1 FW.Furthermore, both mutants also exhibited distinct transcriptional responses among ABA metabolism-, ABA signaling-, dormancy-, and ethylene signaling-related genes.Notably, under high-humidity conditions, sprouting of intact mutant panicles was initially delayed but subsequently exceeded that of WT, reaching approximately 22% in aco2#21-2 and 19% in pi3k#2-3, compared with 13% in WT after 11 days.These findings indicate that ACO2 and PI3K regulate rice seed germination dynamics through distinct modulations in ABA-ethylene homeostasis.Moreover, they demonstrate that enhanced early dormancy-related responses do not necessarily confer sustained PHS resistance during prolonged exposure to humid conditions.