Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence.Levels of ethylene or its precursor 1-aminocyclopropane-1-carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO-associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography-tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.• Our observations showed that the loss-of-function acs1-1 mutant ameliorated age-or dark-induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1-1 reduced ACC accumulation mainly in mature leaves and that acs1-1-promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO-deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.• These findings suggest that NOA1-dependent NO accumulation blocked the ACS1induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.