RESUMO
The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1-OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERF in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl-d-erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila.
RESUMO
The human brain represents an elaborate product of hominizing evolution. Likewise, its supporting vasculature may also embody evolutionary consequences. Thus, it is conceivable that the human tendency to develop cerebral vascular accidents (CVAs) might represent a disease of hominization. In a search for hominizing changes on the arterial circle of Willis (hWAC), we attempted an anatomical comparison of the hWAC with that of the mouse (mWAC) by injecting aliquots of resin into the vasculature of the mouse and then creating vascular endocasts of the mWAC. The internal carotid artery of the mouse (mICA) unites with the mWAC midway between the middle cerebral artery (mMCA) and posterior cerebral artery (mPCA). The mWAC does not complete a circle: the mWAC nourishes the anterior portion of the circle which branches out to the olfactory artery (OlfA) and mPCA, along with the mMCA, and the basilar artery (mBA) does not connect to the mPCA. The OlfA is thicker than the mMCA. The relative brain weight of the mouse was 74 g on average for a 60 kg male and 86 g for a 60 kg female, respectively, as compared with 1424 g for a 60 kg man. These findings are consistent with the mouse being a nocturnal carnivore that lives on olfactory information in contrast to the human that lives diurnally and depends on visual and auditory information. In man, the human ICA (hICA) unites with the hWAC at a point where the human middle cerebral artery (hMCA) branches out, and thus, blood from the hICA does not flow through the hWAC but drains into the hMCA directly. The hMCA is thicker than the anterior cerebral artery. The hPCA receives blood from the hBA rather than from the hICA, and thus, the entire hWAC forms a closed circuit. Since the hICA drains directly into the hMCA without flowing a distance through the hWAC, the capacitor and equalizer functions of the WAC will be mitigated so much that the resultant hemodynamic changes would render the hMCA more likely to contribute to CVAs. Thus, anatomical findings and possibly functions of the arterial circle of Willi may vary from one species to another, depending on one's specific cerebral evolution.
