MicroRNAs (miRNAs) are involved in regulating many aspects of plant growth and development at the post-transcriptional level. Gerbera (Gerbera hybrida) is an important ornamental crop. However, the role of miRNAs in the growth and development of gerbera is still unclear. In this study, we used high-throughput sequencing to analyze the expression profiles of miRNAs in ray floret during inflorescence opening. A total of 164 miRNAs were obtained, comprising 24 conserved miRNAs and 140 novel miRNAs. Ten conserved and 15 novel miRNAs were differentially expressed during ray floret growth, and 607 differentially expressed target genes of these differentially expressed miRNAs were identified using psRNATarget. We performed a comprehensive analysis of the expression profiles of the miRNAs and their targets. The changes in expression of five miRNAs (ghy-miR156, ghy-miR164, ghy-miRn24, ghy-miRn75 and ghy-miRn133) were inversely correlated with the changes in expression of their eight target genes. The miRNA cleavage sites in candidate target gene mRNAs were determined using 5′-RLM-RACE. Several miRNA-mRNA pairs were predicted to regulate ray floret growth and anthocyanin biosynthesis. In conclusion, the results of small RNA sequencing provide valuable information to reveal the mechanisms of miRNA-mediated ray floret growth and anthocyanin accumulation in gerbera.

The CAPRICE (CPC)-like (CPL) genes belong to a single-repeat R3 MYB family, whose roles in physic nut (Jatropha curcas L.), an important energy plant, remain unclear. In this study, we identified a total of six CPL genes (JcCPL1–6) in physic nut. The JcCPL3, 4, and 6 proteins were localized mainly in the nucleus, while proteins JcCPL1, 2, and 5 were localized in both the nucleus and the cytoplasm. Ectopic overexpression of JcCPL1, 2, and 4 in Arabidopsis thaliana resulted in an increase in root hair number and decrease in trichome number. Consistent with the phenotype of reduced anthocyanin in shoots, the expression levels of anthocyanin biosynthesis genes were down-regulated in the shoots of these three transgenic A. thaliana lines. Moreover, we observed that OeJcCPL1, 2, 4 plants attained earlier leaf senescence, especially at the late developmental stage. Consistent with this, the expression levels of several senescence-associated and photosynthesis-related genes were, respectively, up-regulated and down-regulated in leaves. Taken together, our results indicate functional divergence of the six CPL proteins in physic nut. These findings also provide insight into the underlying roles of CPL transcription factors in leaf senescence.

Anthocyanins, biosynthesized through the flavonoid pathway, critically determine floral coloration and ornamental value in plants. While floral development has been extensively studied in Gerbera hybrida, the microRNA-mediated regulation of anthocyanin biosynthesis remains unclear. In this study, we identified and characterized the precursor of gerbera microRNA156 (GhmiR156), which exhibits a typical stem-loop secondary structure. The mature GhmiR156 sequence shows 93.65% similarity with miR156 from other plants. Through target prediction analysis, we identified five potential target genes of GhmiR156, all encoding SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors. Among these, the gene c35442.graph_c0, which shares the highest similarity with AtSPL2 in Arabidopsis, was designated as GhSPL2. Expression analysis revealed an inverse correlation between GhmiR156 and GhSPL2 across different tissues and developmental stages of ray florets. This regulatory relationship was further validated by RLM-5′RACE, which showed that GhmiR156 directly targets and cleaves GhSPL2 mRNA, subsequently supported by dual-luciferase reporter assays and Western blotting analysis. Subcellular localization analysis indicated that GhSPL2 is a nuclear-localized protein, consistent with AtSPL2. Functional analyses revealed that overexpression of GhSPL2 suppressed anthocyanin accumulation by downregulating key biosynthetic genes GhPAL, GhF3H and GhUFGT. Conversely, overexpression of GhmiR156 represses GhSPL2 expression, thereby alleviating its inhibitory effect on anthocyanin accumulation in ray florets, and exhibits an increase in anthocyanin content. Collectively, our findings demonstrate that GhmiR156 fine-tunes the anthocyanin biosynthetic pathway through its target gene GhSPL2. This study provides new insights into the complex regulatory network governing anthocyanin biosynthesis in ornamental plants.