Abstract
Exposure to extended periods of darkness is a common source of abiotic stress that significantly affects plant growth and development. To understand how Nicotiana benthamiana responds to dark stress, the proteomes and metabolomes of leaves treated with darkness were studied. In total, 5763 proteins and 165 primary metabolites were identified following dark treatment. Additionally, the expression of autophagy-related gene (ATG) proteins was transiently upregulated. Weighted gene coexpression network analysis (WGCNA) was utilized to find the protein modules associated with the response to dark stress. A total of four coexpression modules were obtained. The results indicated that heat-shock protein (HSP70), SnRK1-interacting protein 1, 2A phosphatase-associated protein of 46 kDa (Tap46), and glutamate dehydrogenase (GDH) might play crucial roles in N. benthamiana's response to dark stress. Furthermore, a protein-protein interaction (PPI) network was constructed and top-degreed proteins were predicted to identify potential key factors in the response to dark stress. These proteins include isopropylmalate isomerase (IPMI), eukaryotic elongation factor 5A (ELF5A), and ribosomal protein 5A (RPS5A). Finally, metabolic analysis suggested that some amino acids and sugars were involved in the dark-responsive pathways. Thus, these results provide a new avenue for understanding the defensive mechanism against dark stress at the protein and metabolic levels in N. benthamiana.