Leaf senescence, the last stage of leaf development, is a well-regulated and complex process for investigation. For simplification, dark-induced leaf senescence has frequently been used to mimic the natural senescence of leaves because many typical senescence symptoms, such as chlorophyll (Chl) and protein degradation, also occur under darkness. In this study, we compared the phenotypes of leaf senescence that occurred when detached leaves or intact plants were incubated in darkness to induce senescence. We found that the symptoms of non-programmed cell death (non-PCD) with remaining green coloration occurred more heavily in the senescent leaves of whole plants than in the detached leaves. The pheophorbide a (Pheide a) content was also shown to be much higher in senescent leaves when whole plants were incubated in darkness by analyses of leaf Chl and its metabolic intermediates. In addition, more serious non-PCD occurred and more Pheide a accumulated in senescent leaves during dark incubation if the soil used for plant growth contained more water. Under similar conditions, the non-PCD phenotype was alleviated and the accumulation of Pheide a was reduced by overexpressing 7-hydroxymethyl Chl a (HMChl a) reductase (HCAR). Taken together, we conclude that a high soil water content induced non-PCD by decreasing HCAR activity when whole plants were incubated in darkness to induce senescence; thus, the investigation of the fundamental aspects of biochemistry and the regulation of leaf senescence are affected by using dark-induced leaf senescence.
Keywords: Arabidopsis thaliana; HCAR; cell death; chlorophyll degradation; dark-induced leaf senescence.