Catabolic reprogramming of Brassica rapa leaf mesophyll protoplasts during the isolation procedure

The use of leaf mesophyll protoplasts for transformation and genome editing in plants is expected due to the distinctive features of protoplasts, such as cell-wall-free single cells. However, the application of leaf mesophyll protoplasts for molecular breeding is limited because of the recalcitrance of protoplasts to regenerate. We speculated that the primary reason for this recalcitrance is senescence during protoplast isolation before initialization. In this study, we performed profiling, clustering, co-expression analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of primary metabolites and transcriptomes of Brassica rapa leaves and leaf mesophyll protoplasts to reveal the reason for senescence. Primary metabolite profiling indicated that the metabolism of B. rapa protoplasts was converted to catabolism. The number of downregulated differentially expressed genes (DEGs) increased during protoplast isolation. An analysis of GO overexpression revealed the activation of genes involved in catabolic and immune system processes and the inactivation of chloroplasts and photosynthetic genes. KEGG pathway analysis showed that the activation of autophagy and proteasomes accounted for senescence-associated proteolysis during protoplast isolation. It also revealed activation of the genes involved in endoplasmic reticulum stress responses and disease resistance responses during the first 3 h of isolation. These results suggest that elicitor receptor-mediated signal transduction stimulates the pathogen-associated molecular patterns (PAMPs), which triggers immunity. There were more downregulated than upregulated transcription factors during protoplast isolation. AT5G39610 ( ANAC092 , ATNAC2 ) gene expression was significantly activated throughout the entire period of protoplast isolation. ANAC092 was co-expressed with upregulated AT5G26340 ( STP13 ), which encodes a protein with high affinity, hexose-specific/H + symporter. AT1G50030 encodes target of rapamycin ( TOR ) proteins, and the expression of TOR decreased during protoplast isolation. The presence of ethylene and the inhibition of photosynthesis-related genes by glucose and sucrose promote senescence-associated gene expression of protoplasts. Since a decrease in glucose downregulates glucose TOR signaling, the inactivated TOR signaling will promote catabolic reprogramming, senescence, and autophagy in protoplasts during protoplast isolation. We concluded that the initialization of protoplasts requires the blocking of these complicated crosstalk signaling pathways to prevent the catabolic reprogramming of protoplasts, which will lead the way to new plant breeding techniques.