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.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.(AU)

Metabolite fingerprinting of Camptotheca acuminata and the HPLC-ESI-MS/MS analysis of camptothecin and related alkaloids.

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC-MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments.

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkaloid accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

[The study of dynamic acccumulation of camptothecin in Camptotheca acuminata fruit by TLCS].

OBJECTIVE: To establish TLC scanning method for the determination of camptothecin in Camptotheca acuminata fruit, and analysis the dynamic accumulation of camptothecin in Camptotheca acuminata fruit to find out the best collection period. METHODS: Silica gel H-CMC-Na thin layer plate was adopted in the determination with chloroform-acetone (7 : 3) used as deeloper, Single-wavelength and linear scanning of TLC was used, and the detection wavelength was 360 nm. RESULTS: There was a good linear relationship for Comptothecin within the range of 0.0542 - 0.3252 microg, the average recovery was 97.13%, RSD was 1.76%. CONCLUSION: The method is accurate, simple and reliable, and can be used for the determination of camptothecin in Camptotheca acuminata fruit and dynamic accumulation research.

Modelling in vitro growth of dense root networks.

Hairy roots are plants genetically transformed by Agrobacterium rhizogenes, which do not produce shoots and are composed mainly by roots. Hairy roots of Ophiorrhiza mungos Linn. are currently gaining interest of pharmacologists, since a secondary product of their metabolism, camptothecin, is used in chemotherapy. To optimize the production of valuable secondary metabolites it is necessary to understand the metabolism and growth of these roots systems. In this work, a mathematical model for description of apical growth of a dense root network (e.g. hairy roots) is derived. A continuous approach is used to define densities of root tips and root volume. Equations are posed to describe the evolution of these and are coupled to the distribution of nutrient concentration in the medium and inside the network. Following the principles of irreversible thermodynamics, growth velocity is defined as the sum over three different driving forces: nutrient concentration gradients, space gradients and root tip diffusion. A finite volume scheme was used for the simulation and parameters were chosen to fit experimental data from O. mungos Linn. hairy roots. Internal nutrient concentration determines short-term growth. Long-term behavior is limited by the total nutrient amount in the medium. Therefore, mass yield could be increased by guaranteeing a constant supply of nutrients. Increasing the initial mass of inoculation did not result in higher mass yields, since nutrient consumption due to metabolism also rose. Four different growth strategies are compared and their properties discussed. This allowed to understand which strategy might be the best to increase mass production optimally. The model is able to describe very well the temporal evolution of mass increase and nutrient uptake. Our results provide further understanding of growth and density distribution of hairy root network and therefore it is a sound base for future applications to describe, e.g., secondary metabolite production.

CPT accumulation in the fruit and during early phases of plant development in Camptotheca acuminata Decaisne (Nyssaceae).

We describe the dynamics of camptothecin (CPT) accumulation during the early phases of plant development in Camptotheca acuminata. TLC and HPLC analyses were performed on the entire mature fruit and all of its parts and on the organs of seedlings in different developmental stages. In the mature fruit, the CPT content was relatively high (2.83 mg g(-1) DW); it was accumulated especially in the endosperm (1.82 mg g(-1) DW) and embryo (1.10 mg g(-1) DW). Regarding seedlings, CPT was present in all of the organs, at all developmental stages. In the cotyledons, content was greatest in the early developmental stages (4.23 mg g(-1) DW) and drastically decreased once the organ reached maturity (0.34 mg g(-1) DW). A similar trend was observed for the true leaves. The results suggest that CPT is translocated from the organs at an advanced developmental stage to those at an early stage.

[Callus induction and cultivation of Camptotheca acuminata].

OBJECTIVE: To study the callus induction from leaf, stem segments and stem segments with axillary's bud and the subculture conditions of callus in Camptotheca acuminata. METHOD: The explants were inoculated into the media of MS with different concentrations of 6-BA, NAA and 2, 4-D by orthogonal experiment. RESULT AND CONCLUSION: The optimal medium for callus induction was MS with 6-BA 2 mg x L(-1), NAA 2 mg x L(-1) and 2,4-D 2 mg x L(-1). It had better effects in leaf than in other explants. The induction ratio in leaf reached above 80%. The callus subcultured on the medium of MS with 6-BA 1 mg x L(-1) and 2, 4-D 1 mg x L(-1) grew vigorous and more quickly than that in other media. It was loose, friable in consistency and suitable for cell culture.

Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum.

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and terminal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possibility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indicated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (delta-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camptothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camptothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bacterial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.

[Effects of cu2+ on biosynthesis of camptothecin in cell cultures of Camptotheca acuminata].

Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu2+ on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl2 concentration in B5 medium was 0.008 mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu2+ on camptothecin accumulation in light was higher than that in dark.

Molecular characterization of two anthranilate synthase alpha subunit genes in Camptotheca acuminata.

The potent anticancer and antiviral compound camptothecin (CPT) is a monoterpene indole alkaloid produced by Camptotheca acuminata. In order to investigate the biosynthetic pathway of CPT, we studied the early indole pathway, a junction between primary and secondary metabolism, which generates tryptophan for both protein synthesis and indole alkaloid production. We cloned and characterized the alpha subunit of anthranilate synthase (ASA) from Camptotheca (designated CaASA), catalyzing the first committed reaction of the indole pathway. CaASA is encoded by a highly conserved gene family in Camptotheca. The two CaASA genes are differentially regulated. The level of CaASA2 is constitutively low in Camptotheca and was found mainly in the reproductive tissues in transgenic tobacco plants carrying the CaASA2 promoter and beta-glucuronidase gene fusion. CaASA1 was detected to varying degrees in all Camptotheca organs examined and transiently induced to a higher level during seedling development. The spatial and developmental regulation of CaASA1 paralleled that of the previously characterized Camptotheca gene encoding the beta subunit of tryptophan synthase as well as the accumulation of CPT. These data suggest that CaASA1, rather than CaASA2, is responsible for synthesizing precursors for CPT biosynthesis in Camptotheca and that the early indole pathway and CPT biosynthesis are coordinately regulated.

Improvement of growth and camptothecin yield by altering nitrogen source supply in cell suspension cultures of Camptotheca acuminata.

Nitrate at 70 mM gave the highest biomass of Camptotheca acuminata in suspension culture in MS medium, but a NH4+/NO3- molar ratio of 5:1 (giving a total of 40 mM N) gave the maximum camptothecin yield. A two-stage flask culture system was established to improve culture efficiency; cell dry weight, camptothecin content and yield was increased by 30%, 280% and 340%, respectively when compared with those of control, reaching up to 36 g l(-1), 0.36 mg g(-1), and 12.8 mg l(-1), respectively.

[Accumulation and localization of camptothecin in young shoot of Camptotheca acuminata].

The developmental of young stems and leaves were investigated using optical, fluorescence and scanning electron microscopy and the camptothecin content in Camptotheca acuminata was analyzed by HPLC. Secretory structure, especially secretory canals were found. A linear relationship (r(2)=0.9257) was found between CPT content and secretory canal density of stem. The secretion in canals and glandular trichomes showed intense blue autofluorescence under 360 nm UV light. The results showed that camptothecin was primarily accumulated in glandular trichomes and secretory canals of stems and leaves.

[Improved camptothecin production by cell lines of Camptotheca acuminata].

The concentration of camptothecin was determined in different tissues of Camptotheca acuminata seedling. The concentrations of camptothecin in new leaves and roots were significantly higher than in other tissues. However, the concentration of camptothecin declined with leaves becoming old. The induction of callus and cell suspension cultures from younger leaves of Camptotheca acuminata was observed. Cell lines were selected with improved camptothecin production as 0.02%.

Camptothecin accumulation and variations in camptotheca.

Camptotheca (Nyssaceae) is a major source of anticancer camptothecin (CPT). It is imperative to understand CPT accumulation and variations in Camptotheca in order to develop CPT production strategies for endangered germplasm. Our study results showed that CPT is primarily accumulated in glandular trichomes of leaves and stems, and CPT content varies among species and varieties but even more significantly within the plant (with different tissues, tissue ages, and seasons). Because of higher CPT yield and desirable biological and ecological features, 'Hicksii' and 'Katie' should be considered the major management germplasm as CPT sources in the future. Young leaves and mature fruits have higher CPT contents than other tissues in the plants. Young photosynthetic leaves and stems contain higher CPT contents than old ones, but 'sink' tissues such as wood, roots, and fruits show different patterns. CPT content also shows a great seasonal change, but is less influenced by tree age. Intact clipping of young leaves and stems should be managed for harvest for CPT production. Preservation and treatment methods influence the CPT extraction. CPT is better preserved in fresh or freeze-dried material than in air or oven-dried material. CPT can be more efficiently extracted after homogenizer treatment of plant materials because more trichome walls can be broken to allow solvent extraction.