Two classes of cytochrome P450 reductase genes and their divergent functions in Camptotheca acuminata Decne.

Cytochrome P450 monooxygenases (CYP450s) and their auxiliary cytochrome P450 reductases (CPRs) are important and commonly involved in the biosynthesis of camptothecin (CPT). To better understand the possible functions of CPRs in planta, we first isolated two CaCPRs genes from Camptotheca acuminata. Sequence analysis revealed the presence of common conserved FMN-, FAD- and NADPH-binding motifs in putative CaCPR1/CaCPR2 proteins. The two CaCPR paralogs were assigned to the Class I and Class II of CPRs, respectively, according to phylogenetic tree. The recombinant CrCYP72-CaCPR1 and CrCYP72-CaCPR2 enzymes and their substrate bioconversion rates of 23.09% and 35.23% demonstrated that both CaCPRs could support the enzyme activities of CrSLS1. Gene silencing of CaCPRs by VIGS led to downregulation of CaCPR1/CaCPR2 expression by 50-67%, accompanied with 10-15% slight decrease and 57-63% dramatic reduction for CaCPR1 and CaCPR2 individually in CPT accumulations. Moreover, CaCPR1/CaCPR2 displayed almost omnipresent expression patterns across Camptotheca tissues. While in comparison to constitutive expression of CaCPR1 gene, CaCPR2 and CYP450 genes were all dramatically phytohormone-induced expressed in leaves which were main tissues for isoprenoid and CPT biosynthesis. Our results suggested that, in Camptotheca seedlings, CaCPR2 had a distinct function from CaCPR1 that was clearly involved in the inducible specialized metabolism for CPT biosynthesis.

Floral morphology and morphogenesis in Camptotheca (Nyssaceae), and its systematic significance.

Background and Aims: Camptotheca is endemic to China and there are limited data about the breeding system and morphogenesis of the flowers. Camptotheca is thought to be related to Nyssa and Davidia in Nyssaceae, which has sometimes been included in Cornaceae. However, molecular phylogenetic studies confirmed the inclusion of Camptotheca in Nyssaceae and its exclusion from Cornaceae. The aim of this study was to reveal developmental features of the inflorescence and flowers in Camptotheca to compare with related taxa in Cornales. Methods: Inflorescences and flowers of Camptotheca acuminata at all developmental stages were collected and studied with a scanning electron microscope and stereo microscope. Key Results: Camptotheca has botryoids which are composed of several capitate floral units (FUs) that are initiated acropetally. On each FU, flowers are grouped in dyads that are initiated acropetally. All floral organs are initiated centripetally. Calyx lobes are restricted to five teeth. The hypanthium, with five toothed calyx lobes, is adnate to the ovary. The five petals are free and valvate. Ten stamens are inserted in two whorls around the central depression, in which the style is immersed. Three carpels are initiated independently but the ovary is syncarpous and unilocular. The ovule is unitegmic and heterotropous. Inflorescences are functionally andromonoecious varying with the position of the FUs on the inflorescence system. Flowers on the upper FU often have robust styles and fully developed ovules. Flowers on the lower FU have undeveloped styles and aborted ovules, and the flowers on the middle FU are transitional. Conclusions: Camptotheca possesses several traits that unify it with Nyssa, Mastixia and Diplopanax. Inflorescence and floral characters support a close relationship with Nyssaceae and Mastixiaceae but a distant relationship with Cornus. Our results corroborate molecular inferences and support a separate family Nyssaceae.

De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin.

Camptotheca acuminata is 1 of a limited number of species that produce camptothecin, a pentacyclic quinoline alkaloid with anti-cancer activity due to its ability to inhibit DNA topoisomerase. While transcriptome studies have been performed previously with various camptothecin-producing species, no genome sequence for a camptothecin-producing species is available to date. We generated a high-quality de novo genome assembly for C. acuminata representing 403 174 860 bp on 1394 scaffolds with an N50 scaffold size of 1752 kbp. Quality assessments of the assembly revealed robust representation of the genome sequence including genic regions. Using a novel genome annotation method, we annotated 31 825 genes encoding 40 332 gene models. Based on sequence identity and orthology with validated genes from Catharanthus roseus as well as Pfam searches, we identified candidate orthologs for genes potentially involved in camptothecin biosynthesis. Extensive gene duplication including tandem duplication was widespread in the C. acuminata genome, with 2571 genes belonging to 997 tandem duplicated gene clusters. To our knowledge, this is the first genome sequence for a camptothecin-producing species, and access to the C. acuminata genome will permit not only discovery of genes encoding the camptothecin biosynthetic pathway but also reagents that can be used for heterologous expression of camptothecin and camptothecin analogs with novel pharmaceutical applications.