MicroRNAs (miRNAs) are small noncoding RNAs that guide plant development by reducing the activity of specific target genes. microRNA 396 (miR396) is an ancient and widely conserved miRNA that classically regulates GROWTH-REGULATING FACTOR (GRF) transcription factor genes, which influence cell proliferation, organ size, and responses to developmental and environmental signals. However, miR396 can also acquire lineage-specific targets, suggesting that plant genomes continually reshape old regulatory circuits for new functions. How these new miRNA–target relationships arise remains a key question in evolutionary biology. Based on these challenges, deeper research is needed into how synonymous substitutions create or disrupt miRNA targeting during plant genome evolution.
A team from the College of Horticulture, Zhejiang Agriculture and Forestry University reported (DOI: 10.1093/hr/uhag036) the findings in Horticulture Research on 16 February 2026. The article identifies CsaWPRa4, which encodes a WEB1 (Weak Chloroplast Movement under Blue Light 1)/PMI2 (Plastid Movement Impaired 2)-related protein (WPR), as a newly evolved target of cucumber miR396. The study combines comparative sequence analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), luciferase assays, tobacco ringspot virus (TRSV)-based silencing, gene overexpression, RNA sequencing (RNA-seq), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing.
The researchers first predicted CsaWPRa4 as a candidate miR396 target and found that its miR396 binding site lies within the coding sequence for the conserved AAKKAVE motif. Although the WPRa4 protein is highly conserved across plants, the RNA sequence encoding this motif differs among species. In cucumber and several cucurbit relatives, synonymous substitutions improved sequence pairing with miR396, whereas WPRa4 homologs in Arabidopsis, tomato, soybean, and rice lacked comparable targeting potential. The team then confirmed negative regulation of CsaWPRa4 by miR396 through multiple lines of evidence: older cucumber leaves showed higher CsaMIR396 expression and lower CsaWPRa4 expression; luciferase activity decreased when CsaMIR396A was co-expressed with the CsaWPRa4 target site; TRSV-mediated miR396 silencing increased CsaWPRa4 expression; and CsaMIR396A overexpression reduced CsaWPRa4 expression. CRISPR/Cas9-generated Csawpra4 mutants further showed altered chlorophyll content, photosynthetic rate, and flower morphology, including abnormal flowers with changed petal and sepal numbers.
The authors said the study highlights a hidden layer of genome evolution: synonymous substitutions can preserve a protein while changing how its messenger RNA is regulated. They said the miR396–CsaWPRa4 pathway provides a clear example of how a conserved gene can be recruited into a new regulatory network through small coding-sequence changes. They also said this mechanism may help explain how plant lineages develop new physiological and developmental features without disrupting essential protein functions.
These findings expand the biological meaning of synonymous substitutions in crops. Rather than being treated only as neutral background variation, such changes may mark hidden regulatory switches that affect photosynthesis, flowering, growth, and adaptation. For cucumber and other cucurbits, the miR396–CsaWPRa4 module offers a new entry point for studying how developmental and environmental signals are translated into visible traits. More broadly, the study provides a framework for scanning crop genomes for miRNA binding sites that have been gained or lost through synonymous substitutions. Such knowledge could eventually support more precise breeding strategies that target gene regulation without altering protein structure.
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References
DOI
10.1093/hr/uhag036
Original Source URL
https://doi.org/10.1093/hr/uhag036
Funding information
This work was supported by the Natural Science Foundation of Hangzhou City (2025SZRJJ0025), the Major Project of Science and Technology Innovation 2025 of Ningbo (2021Z006) and the Key Science Project of Vegetable Breeding in Zhejiang (2021C02065).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.