Competition Binding of CmbHLH1L and CmNLP6/7L to CmbHLH63 Modulates Leaf Senescence in Chrysanthemum

Leaf senescence impacts the quality, productivity and post-harvest performance of crops and ornamental plants. However, the underlying molecular mechanisms governing this process remain poorly understood. In this study, we identified CmbHLH63 as a 'brake signal' of leaf senescence in chrysanthemum and elucidated its critical regulatory role within the CmbHLH1L-CmbHLH63-CmNLP6/7L complex. CmbHLH63 promotes leaf senescence, while it is downregulated during this process. Notably, CmbHLH63 lacks intrinsic transcriptional regulatory activity; however, the expression of a majority of genes was repressed in plants overexpressing CmbHLH63. Further investigation revealed that this repression is achieved through recruiting the transcriptional repressor CmbHLH1L. We further found that CmNLP6/7L, a transcriptional activator, competes with CmbHLH1L for the interaction with CmbHLH63, leading to the release of their inhibition on downstream gene CmNLP6/7L. We found that the accumulation of CmbHLH1L is increased in the cytoplasm and decreased in the nucleus during leaf senescence. Mass spectrometry assays and subcellular localization studies revealed that CmbHLH1L undergoes lysine acetylation at position 140, which prevents its nuclear accumulation, thereby elevating CmNLP6/7L expression during leaf senescence.