The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions.

Cytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6-, the putative mutant copy with a 300 bp truncation at the 5'-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6+) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6+ copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6+ is expressed only in MF samples, while apt6- mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6- could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility.

Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator.

Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).