Leaf chlorosis and senescence are key indicators of prolonged drought stress. In this study, we found that suppressing the chlorophyll b reductase gene (LpNOL) delayed drought-induced leaf chlorosis in perennial ryegrass (Lolium perenne). Through a yeast one-hybrid (Y1H) library screen, we identified a NAC transcription factor, designated chlorophyll b degradation regulator 1 (LpCbDR1), as a direct activator of LpNOL. Subcellular localization analysis confirmed that LpCbDR1 localizes to the nucleus, and its direct binding to the LpNOL promoter was validated by electrophoretic mobility shift assay (EMSA) and CUT&Tag-qPCR assays. Overexpression of LpCbDR1 accelerated leaf senescence, whereas knockdown of LpCbDR1 delayed leaf senescence. Notably, LpCbDR1’s expression was not only upregulated during leaf senescence but also induced by osmotic stress, promoting further investigation into its role and underlying mechanisms in regulating drought tolerance. Phenotypic analysis showed that LpCbDR1-overexpressing lines exhibited significantly higher drought tolerance compared to wild-type (WT) plants, while LpCbDR1-RNAi lines were drought-sensitive than WT. Integrated RNA-seq and CUT&Tag analysis identified LpPLA7 and LpERF1B as downstream targets of LpCbDR1. Directly binding of LpCbDR1 to the promoter of LpPLA7 and LpERF1B was confirmed by Y1H, EMSA, and CUT&Tag-qPCR assays. Both LpPLA7 and LpERF1B were drought-inducible, and functional validation revealed that overexpression of either gene enhanced osmotic stress tolerance in both WT and LpCbDR1-RNAi backgrounds. Collectively, this study demonstrates that LpCbDR1 regulates natural, dark-, and drought-induced leaf senescence by activating LpNOL, and improves drought tolerance at least partially through direct activation of LpPLA7 and LpERF1B in perennial ryegrass.

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