Abstract: Programmed cell death (PCD) is essential for animal and plant development. However, the knowledge of the mechanism regulating PCD in plants remains limited, largely due to technical limitations. Previously, we determined that the protease NtCP14 could trigger PCD in the embryonic suspensor of tobacco (Nicotiana tabacum), providing a unique opportunity to overcome the limitations by creating synchronous two-celled proembryos with ongoing PCD for transcriptome analysis and regulatory factor screening. Here, we performed comparative transcriptome analysis using isolated two-celled proembryos and explored the potential regulatory network underlying NtCP14-triggered PCD. Multiple phytohormones, calcium, microtubule organization, the immunity system, soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins, long non-coding RNAs and alternative splicing are addressed as critical factors involved in the early stage of suspensor PCD. Genes thought to play crucial roles in suspensor PCD are highlighted. Notably, decreased antioxidant gene expression and increased reactive oxygen species (ROS) levels during suspensor PCD suggest a critical role for ROS signaling in the initiation of NtCP14-triggered PCD. Furthermore, five genes in the regulatory network are recommended as immediate downstream elements of NtCP14. Together, our analysis outlines an overall molecular network underlying protease-triggered PCD and provides a reliable database and valuable clues for targeting elements immediately downstream of NtCP14 to overcome technical bottlenecks and gain deep insight into the molecular mechanism regulating plant PCD.

Key words: programmed cell death, protease, reactive oxygen species, suspensor, transcriptome