miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

BACKGROUND: Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. RESULTS: Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. CONCLUSIONS: We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Lignan Constituents from the Fruits of Viburnum macrocephalum f. keteleeri and Their α-Amylase, α-Glucosidase, and Protein Tyrosine Phosphatase 1B Inhibitory Activities.

Eight previously undescribed lignan glycosides, viburmacrosides A-H (1-8), and seven known analogues (9-15) were isolated from Viburnum macrocephalum f. keteleeri fruits through bioactivity-guided fractionation. Their structures and absolute configurations were elucidated by extensive spectroscopic analyses and chemical evidence. Using the well-recognized carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase, as well as the promising protein tyrosine phosphatase 1B (PTP1B), as inhibitory targets, all isolated compounds were tested for their antidiabetic potential in vitro. Compound 4 displayed potent inhibitory activities with IC50 values of 9.9 ± 0.6 and 8.9 ± 0.5 µM against α-glucosidase and PTP1B, respectively. The enzymatic kinetics results suggested that compound 4 competitively inhibited α-glucosidase while it suppressed α-amylase and PTP1B in the mixed-type manner. These findings supported that V. macrocephalum f. keteleeri fruits may be a new functional food resource with antidiabetic potential.

Transcriptomic Analysis Reveals Mechanisms of Sterile and Fertile Flower Differentiation and Development in Viburnum macrocephalum f. keteleeri.

Sterile and fertile flowers are an important evolutionary developmental (evo-devo) phenotype in angiosperm flowers, playing important roles in pollinator attraction and sexual reproductive success. However, the gene regulatory mechanisms underlying fertile and sterile flower differentiation and development remain largely unknown. Viburnum macrocephalum f. keteleeri, which possesses fertile and sterile flowers in a single inflorescence, is a useful candidate species for investigating the regulatory networks in differentiation and development. We developed a de novo-assembled flower reference transcriptome. Using RNA sequencing (RNA-seq), we compared the expression patterns of fertile and sterile flowers isolated from the same inflorescence over its rapid developmental stages. The flower reference transcriptome consisted of 105,683 non-redundant transcripts, of which 5,675 transcripts showed significant differential expression between fertile and sterile flowers. Combined with morphological and cytological changes between fertile and sterile flowers, we identified expression changes of many genes potentially involved in reproductive processes, phytohormone signaling, and cell proliferation and expansion using RNA-seq and qRT-PCR. In particular, many transcription factors (TFs), including MADS-box family members and ABCDE-class genes, were identified, and expression changes in TFs involved in multiple functions were analyzed and highlighted to determine their roles in regulating fertile and sterile flower differentiation and development. Our large-scale transcriptional analysis of fertile and sterile flowers revealed the dynamics of transcriptional networks and potentially key components in regulating differentiation and development of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri. Our data provide a useful resource for Viburnum transcriptional research and offer insights into gene regulation of differentiation of diverse evo-devo processes in flowers.