As an evolutionarily conserved microRNA (miRNA), miR396 regulates plant growth by integrating developmental and environmental signals. In the present study, CsaWPRa4, a WEB1 (Weak Chloroplast Movement under Blue Light 1)/PMI2 (Plastid Movement Impaired 2)-related protein (WPR) family member, was predicted to be a novel target gene of CsamiR396 in cucumbers. WPRa4 is a highly conserved protein in plants. Interestingly, bioinformatic analysis showed that WPRa4 acts as a conserved target gene of miR396 in cucumber and its related species in cucurbits, but not in other plants. The miR396 binding site is located within the coding region of the AAK(K/R)AVE motif in WPRa4, and it evolved by synonymous substitutions in cucurbits. Negative regulation of CsaWPRa4 by CsamiR396 was confirmed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), luciferase assay, gene overexpression, and tobacco ringspot virus (TRSV)-based gene silencing analysis. The subcellular localization assay showed that CsaWPRa4 was localized to both the cell periphery and nuclear periphery. Thereafter, Csawpra4 mutants were generated using CRISPR/Cas9-mediated gene editing. Chloroplast- and flower morphogenesis-related genes were altered, resulting in altered photosynthetic traits and flower morphogenesis in Csawpra4 mutants. In summary, our results showed that WPRa4 evolved as a novel target of miR396 through synonymous substitutions in cucurbits, uncovering the role of synonymous substitutions in genome evolution and providing a new perspective on miRNA–target evolutionary processes in plants.

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