As an important source of human dietary nutrition, the development and quality formation of fruits from horticultural crops are fine-tuned by sophisticated genetic networks. In the past several years, along with the rapid development of molecular biology and functional genetics, microRNAs (miRNAs), a representative class of non-coding RNAs, have emerged as key post-transcriptional regulators, playing an increasingly prominent role in fruit development and quality control. In this review, the multiple regulatory functions of miRNAs are systematically elucidated during fruit development. Firstly, a brief introduction about the conserved biosynthetic pathways of plant miRNAs and their expression profiles in fruits are provided. Subsequently, the core regulatory modules and functional programs of miRNAs are well described by following the developmental stages, mainly including fruit setting and early initiation, fruit enlargement, ripening-associated coloration and softening, and quality formation. In particular, the key modules such as miR156/157-SPL, miR172-AP2, miR396-GRF, miR397-LAC, miR828/858-MYB, miR159-GAMYB2 and miR164-NAC are highlighted to regulate fruit size and morphological shaping, the timing of ripening, the formation of color and flavor, cell-wall remodeling, the maintenance of shelf life, and so on. Moreover, the action features of miRNAs, mainly via cooperating mineral homeostasis, hormone signaling, reactive oxygen species pathways, are discussed in modulating fruit development and quality under different stressful conditions. The potential application of miRNA-involved strategies, including gene editing, short tandem target mimics (STTM) and tissue-specific expression, is also evaluated in molecular breeding, while simultaneously challenges are highlighted in current miRNA research about functional validation, regulatory network dissection and breeding adoption. Cutting-edge technologies in this field are finally proposed, emphasizing the integration of multi-omics, single-cell sequencing, and artificial intelligence to systematically decipher miRNA regulatory networks and advance the molecular design breeding of fruit traits. Altogether, this review provides a comprehensive theoretical reference for mechanistic understanding of miRNA-mediated regulation in fruit development and its future utilization in genetic enhancement of horticultural crops.
