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. 2023 Mar 14;35(6):2271–2292. doi: 10.1093/plcell/koad077

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© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — PpRAP2.4 positively regulates anthocyanin and ethylene biosynthesis. A) Overexpression of PpRAP2.4 slightly induced the anthocyanin accumulation in pear calli. The calli transformed with the empty vector (pCAMBIA1301) were used as the negative control. Pear calli were incubated under strong light at 17°C for 5 d. B) Anthocyanin contents in PpRAP2.4-OX pear calli after light treatment. C) Expression levels of anthocyanin-related genes and PpRAP2.4 in PpRAP2.4-OX pear calli after light treatment. D) Relative growth rate of PpRAP2.4-OX and control pear calli over 2 wk. E) Ethylene releasing rate of PpRAP2.4-OX and control pear calli. F) Expression levels of key ethylene biosynthetic genes (PpACS1 and PpACO1) and PpERF9 in PpRAP2.4-OX and control pear calli. G) A Y1H assay revealed that PpRAP2.4 can bind directly to the PpACS1 promoter. H) An EMSA revealed that PpRAP2.4 can bind directly to the DRE motif of the PpACS1 promoter. The hot probe was a biotin-labeled fragment of the PpACS1 promoter containing the DRE motif, whereas the competitor probe was an unlabeled probe (50-, 100-, and 200-fold molar excess). The mutant cold probe was the same as the labeled hot probe but with CCGAC mutated to TTTTT. The His-tagged PpRAP2.4 protein was purified. I) A dual-luciferase assay indicated that PpRAP2.4 activates the PpACS1 promoter in vivo. The promoter of PpACS1 was cloned into the pGreenII 0800-LUC (firefly luciferase) vector, and the full-length CDS of PpRAP2.4 was cloned into the pGreenII 0029 62-SK vector. The empty vector of pGreenII 0029 62-SK was used as control. The relative luciferase activity was analyzed. J and L) The 1-MCP treatment significantly induced the J) red coloration and increased the L) total anthocyanin content in PpRAP2.4-OX pear calli. K) The 1-MCP treatment decreased the ethylene release rate in PpRAP2.4-OX and control pear calli. M) Expression levels of anthocyanin-related genes and PpRAP2.4 in PpRAP2.4-OX pear calli after the 1-MCP treatment. N to P) Silencing of PpRAP2.4 inhibited the red coloration N), ethylene releasing rate O), and total anthocyanin contents P) in pear calli. Q) Expression levels of PpRAP2.4 and anthocyanin-related genes in PpRAP2.4-RNAi pear calli and control pear calli (empty vector) after the light treatment. Error bars represent the standard deviation of 3 biological replicates. Asterisks indicate significantly different values (*P < 0.05 and **P < 0.01).