Abstract
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.
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Supplementary file1 (PDF 1070 kb) Temporal changes in gene expression from leaves to protoplasts. A total of 16,386 DEGs were clustered by MeV_4_8 (v.10.2) based on the k-means method (k = 9). When starting MeV, main menu bar and viewer are opened. From viewer menu, selected Load Data in File menu bar. Expression File Loader window appears. Select txt file (Supplementary Table 2, log2cpm_tmm_norm) from Browse button. Mark Single-Color Array button and load data. Select K-Means/Medians Clustering in Clustering menu bar, KMC: K-Means/K-Medians window appears. Select Pearson Correlation, mark Calculate K-Means and set 9 for number of clusters, then click OK. Click arrow for Analysis Results, KMC-genes, and Expression Graphs in Analysis Result window on the left side of viewer, and the results of k-means clustering graphs appear. Select All Cluster graph and save as a TIFF file.
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Supplementary file2 (PDF 880 kb) GO overrepresentation analysis of induction and repression subcategories in MF category. Analyses were performed by clusterProfiler R package (Yu et al. 2012). A self-made function was created in R with the clusterProfiler R package. In short, this function performs the following steps: The files to read are the differentially expressed genes in Supplementary Table 1, DE_Leaf3h_vs_Leaf0h/DE_Protoplast_vs_Leaf0h/DE_Protoplast_vs_Leaf3h files. TAIR ID was converted to ENTREZ ID with bitr command of R. According to the clusterProfiler manual, AdjustMethod = "BH", pvalueCutoff = 0.01, and qvalueCutoff = 0.05 were set as enrichGO command.
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Supplementary file3 (PDF 3266 kb) Expression profiles of metabolic pathways based on KEGG pathway analysis. 3.1 (a) pentose phosphate pathway, (b) citrate cycle, (c) oxidative phosphorylation; 3.2 (a) endocytosis, (b) soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) interactions in vesicular transport, (c) ubiquitin mediated proteolysis, (d) protein processing in endoplasmic reticulum. Expression profiles of all genes and metabolites are color coded in each pathway with data from each time point in the same order as Figure 5. A self-made function was created in R with pathview and imager packages. In short, this function performs the following steps: The files to read are Supplemental Table 1, All_EdgeR.LogData for gene expression data and Supplementary Table 5, Primary metabolites for primary metabolite expression data. Genes from the KEGG pathway were combined with gene expression data. Genes listed in the KEGG pathway map of interest were obtained by the terminal command, and the absolute value of their gene expression was calculated to display the color code of the fold change. KEGG gene expression data and primary metabolite data (Supplementary Table 5, Primary metabolites) were converted into a matrix for reading in R pathview package. Upper limit of gene expression was set to absolute value of those gene expressions. Upper limit of primary metabolites was set to 1 by default. According to the R pathview manual, genes and primary metabolite expression matrix, KEGG pathway ID, integers of absolute values of those gene expressions, etc., were set as R pathview commands. Since the package outputs a PNG file, the file was converted to a PDF by the imager package.
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Supplementary file4 (PDF 38 kb) Heat map showing expression levels of 295 differentially expressed transcription factor genes. Red indicates relatively high expression and blue relatively low expression. A self-made function was created in R with ggplot2 and other packages. In short, this function performs the following steps: The files to read are the transcription factor expression data file (Supplementary Table 6, Expression data of TF) and gene expression data with a CPM value higher than 0.5 (Supplementary Table 2, log2cpm_tmm_norm). Transcription factor genes showing expression levels greater than log2 > ± 1 were extracted by comparing the two treatment libraries and combining with gene expression data with CPM values greater than 0.5. The created data frame was converted to a matrix and the R heatmap command was executed for clustering. The data were sorted according to the clustering result. Finally, geom_tile of the ggplot2 package was used to finish the heatmap.
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Supplementary file5 (PDF 906 kb) Co-expression of differentially expressed inducible transcription factor genes extracted from overlapping Venn diagram of Leaf0h, Leaf3h, and Protoplast and their first direct neighbors analyzed in Cytoscape (cytoscape.org). Ensembl gene IDs of differentially expressed transcription factor genes listed in Supplementary Table 1, Prot_Leaf3h_Leaf0h_EdgR_UP_TFac were imported into the obtained network. ANAC092 (AT5G39610) was selected in the network and its first neighbors of selected nodes were only selected by hiding unselected nodes and edges. All edges were concealed by unchecking all networks on the Networks tab on the right. yFiles Orthogonal Layout was selected in the Layout menu. Clicking the Filter tab on the left, the Column Filter was set as follows; L3h.L0h_logFC, P.L0h_logFC, and P.L3h_logFC is not between -1 and 1 inclusive for Leaf3h vs. Leaf0h, Protoplast vs. Leaf0h, and Protoplast vs. Leaf3h, respectively. Hide Selected Nodes and Edges was selected in the Select menu bar. Default style was selected on the Style tab on the left. Clicking the Fill Color pull-down menu brings up the Column, Mapping Type, and Current Mapping submenu. In the Column menu, L3h.L0h_logFC was selected for Leaf3h vs. Leaf0h, P.L0h_logFC was selected for Protoplast vs. Leaf0h, and P.L3h_logFC was selected for Protoplast vs. Leaf3h. Continuous Mapping was selected in Mapping Type menu. Double-clicking the color bar in Current Mapping launches Continuous Mapping Editor for Node Fill Color window. When L3h.L0h_logFC was selected in the Column comparing Leaf3h and Leaf0h, the Set Min and Max button was clicked. Double-clicking the lowest (-8.31) and highest (11.25) limit marker launches the Colors window. The Sample(S) color code table was selected by clicking the arrow in the Colors window menu. Blue (0, 0, 255) and Red (255, 0, 0) were selected for the lowest and highest limit, respectively. Arrow in center was clicked, and the handle position button was adjusted to zero. Clicking the Node Fill Color button launches the Colors window. White (255, 255, 255) was selected for the center point. The Export as Image button on the bottom of the main window was selected, and the images were saved as PDF files.
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Supplementary file6 (PDF 1014 kb) Co-expression of hexose transporter STP13 gene and differentially expressed transcription factor genes. Co-expression of STP13 transcription factor gene and first direct neighbor genes analyzed by Cytoscape (cytoscape.org). Ensembl gene IDs of differentially expressed transcription factor genes listed in Supplementary Table 6, TF Expression Data and STP13 (AT5G26340) were imported into obtained network. STP13 (AT5G26340) was selected in the network and its first neighbors of selected nodes were only selected by hiding unselected nodes and edges. Subsequent procedures were the same as in Supplementary Figure 5. Grid layout was selected in the layout menu.
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Supplementary file7 (PDF 1119 kb) Co-expression analysis of AT3G11020 (DREB2B), a member of DREB subfamily in A-2 of ERF/AP2 transcription factor family and differentially expressed transcription factor genes. Co-expression analysis was performed by Cytoscape (cytoscape.org). Ensembl gene IDs of differentially expressed transcription factor genes listed in Supplementary Table 6, TF Expression Data were imported into the obtained network. AT3G11020 (DREB2B ) was selected in the network and its first neighbors of selected nodes were only selected by hiding unselected nodes and edges. Subsequent procedures were the same as in Supplementary Figure 5. Grid layout was selected in the layout menu.
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Nakayama, Y., Kusano, M., Kobayashi, M. et al. Catabolic reprogramming of Brassica rapa leaf mesophyll protoplasts during the isolation procedure. Plant Growth Regul 99, 337–357 (2023). https://doi.org/10.1007/s10725-022-00912-y
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DOI: https://doi.org/10.1007/s10725-022-00912-y