ScRNA-seq reveals the spatiotemporal distribution of camptothecin pathway and transposon activity in Camptotheca acuminata shoot apexes and leaves.

Camptotheca acuminata Decne., a significant natural source of the anticancer drug camptothecin (CPT), synthesizes CPT through the monoterpene indole alkaloid (MIA) pathway. In this study, we used single-cell RNA sequencing (scRNA-seq) to generate datasets encompassing over 60,000 cells from C. acuminata shoot apexes and leaves. After cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Analysis of MIA pathway gene expression revealed that most of them exhibited heightened expression in proliferating cells (PCs) and vascular cells (VCs). In contrast to MIA biosynthesis in Catharanthus roseus, CPT biosynthesis in C. acuminata did not exhibit multicellular compartmentalization. Some putative genes encoding enzymes and transcription factors (TFs) related to the biosynthesis of CPT and its derivatives were identified through co-expression analysis. These include 19 cytochrome P450 genes, 8 O-methyltransferase (OMT) genes, and 62 TFs. Additionally, these pathway genes exhibited dynamic expression patterns during VC and EC development. Furthermore, by integrating gene and transposable element (TE) expression data, we constructed novel single-cell transcriptome atlases for C. acuminata. This approach significantly facilitated the identification of rare cell types, including peripheral zone cells (PZs). Some TE families displayed cell type specific, tissue specific, or developmental stage-specific expression patterns, suggesting crucial roles for these TEs in cell differentiation and development. Overall, this study not only provides novel insights into CPT biosynthesis and spatial-temporal TE expression characteristics in C. acuminata, but also serves as a valuable resource for further comprehensive investigations into the development and physiology of this species.

Biotechnological approaches for the production of camptothecin.

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved . KEY POINTS: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production.

Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in Camptotheca.

Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

Integrated transcriptome and proteome analysis provides new insights into camptothecin biosynthesis and regulation in Camptotheca acuminata.

Camptotheca acuminata Decne., the main source of camptothecin (CPT), has received increasing attention for its remarkable antitumor activity. Many CPT derivatives are clinically used as effective anticancer agents worldwide. However, their biosynthesis mechanism remains unclear, and uncovering this pathway would greatly facilitate development of alternative CPT production methods to replace current inefficient plant-derived ones. The expression of >30,000 genes was accurately quantified using unique molecular identifier RNA sequencing in 10 C. acuminata tissues, and 7854 proteins from five tissues were quantified with label-free quantitative proteomics. Fifteen full-length transcriptomes were sequenced with long-read Oxford Nanopore Technologies, and 5692 alternative splicing events were discovered among 4746 genes. Integrated transcriptome and proteome analysis provided novel insights into CPT biosynthesis and its hierarchical regulation. Five cytochrome P450s and three O-methyltransferases were considered as candidates involved in the biosynthesis of CPT and its derivatives, while 15 transcription factors potentially regulating CPT biosynthesis were screened. These findings provide important clues for elucidating the biosynthetic mechanisms of CPT and its derivatives and substantially contribute to the future production of these anticancer agents with synthetic biology. The generated large-scale multiomics data also provide valuable resources for investigating the functional genomics of the most important CPT-producing plant species-C. acuminata.

Phytochemical Analysis of Nothapodytes tomentosa and Distribution and Content of Camptothecin and its Analogues in Four Plants.

Camptothecin (CPT) and its derivatives have attracted worldwide attention because of their notable anticancer activity. However, the growing demand for CPT in the global pharmaceutical industry has caused a severe shortage of CPT-producing plant resources. In this study, phytochemical analysis of Nothapodytes tomentosa results in the isolation and identification of CPT (13: ) and 16 analogues (1:  - 12, 14:  - 17: ), including a new (1: ) and five known (9, 10, 12, 15: , and 17: ) CPT analogues with an open E-ring. In view of the potential anticancer activity of CPT analogues with an open E-ring, the fragmentation pathways and mass spectra profiles of these six CPT analogues (1, 9, 10, 12, 15: , and 17: ) are investigated, providing a reference for the rapid detection of these compounds in other plants. Furthermore, based on the fragmentation patterns of CPT (13: ) and known analogues (2:  - 8, 11, 14, 16, 18:  - 26: ), the distribution and content of these compounds in different tissues of N. tomentosa, N. nimmoniana, Camptotheca acuminata, and Ophiorrhiza japonica are further studied. Our findings not only provide an alternative plant resource for further expanding the development and utilization of CPT and its analogues, but also lay a foundation for improving the utilization of known CPT-producing plant resources.

Biotechnology for micropropagation and camptothecin production in Ophiorrhiza sp.

Camptothecin (CPT) is a monoterpenoid-alkaloid, an anticancer compound from plant. Ever since its discovery in 1996 from the bark of Camptotheca acuminata, various researches have been conducted for enhancing its production. CPT has also been reported in several other species belonging to the plant families Icacinaceae, Rubiaceae, Apocynaceae, Nyssaceae, Betulaceae, Violaceae, Meliaceae, and Gelseminaceae. Out of these, Ophiorrhiza sp. (Rubiaceae) is the next possible candidate for sustainable CPT production after C. acuminata and Nothapodytes nimoonia. Various biotechnological-studies have been conducted on Ophiorrhiza sp. for searching the elite species and the most optimal strategies for CPT production. The genus Ophiorrhiza has been used as medicines for antiviral, antifungal, antimalarial, and anticancer activities. Phytochemical analysis has revealed the presence of alkaloids, flavonoids, triterpenes, and CPT from the plant. Because of the presence of CPT and its herbaceous habit, Ophiorrhiza sp. has now become a hot topic in research area. Currently, for mass production of the elite spp., tissue culture techniques have been implemented. In the past decades, several researchers have contributed on the diversity assessment, phytochemical analysis, mass production, and in vitro production of CPT in Ophiorrhiza sp. In this paper, we review the on the biotechnological strategies, optimal culture medium, micropropagation of Ophiorrhiza sp., effect of PGR on shoot formation, rhizogenesis, callus formation, and enhanced production of CPT for commercial use. KEY POINTS: • Latest literature on in vitro propagation of Ophiorrhiza sp. • Biotechnological production of camptothecin and related compounds • Optimization, elicitation, and transgenic studies in Ophiorrhiza sp.

Valuable alkaloids content is preserved in Camptotheca acuminata and Morus alba grown in trace elements contaminated soil.

Phytoextraction of trace elements (TE) using woody species is an economically challenging soil remediation approach because of the long time needed. Yet, some trees contain alkaloids that can be exploited along structural components to enhance biomass value. As alkaloids are thought to be involved in plant defence mechanisms, we hypothesized that potentially hostile phytoremediation conditions could increase their level. Camptothecin in Camptotheca acuminata and 1-deoxynojirimycin in Morus alba were measured from trees grown in a field in presence of Cu, Pb and Zn all together, and from M. alba grown in a greenhouse in presence of Cd or other abiotic stressors (NaCl and bending). The trees did not extract TE in the field, but M. alba stems accumulated Cd in the greenhouse experiment, with no consequence on stomatal conductance and leaves pigments concentration. Camptothecin and 1-deoxynojirimycin concentrations were preserved under all experimental conditions, as was biomass yield, and phenolics were slightly increased in M. alba exposed to TE. This study provides evidence that valuable and persistent alkaloids and phenolics can be extracted from trees facing phytoremediation-associated stresses, without a negative impact on their quantity and on biomass yield. Such products could generate a sustainable stream of revenues during phytoremediation.

There is scarce data on tree alkaloid content and scarcer data on how it is affected by exposure to trace elements in a phytoremediation context. We provide evidence that the content of two specific alkaloids is not altered in Morus alba and Camptotheca acuminata exposed to moderate to elevated levels of contaminating trace elements. The manuscript introduces the use of M. alba for phytoremediation in the Americas and is the first to propose the use of C. acuminata on trace element contaminated sites to produce camptothecin, a valuable anticancer alkaloid.

An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

Penpolonin A-E, cytotoxic α-pyrone derivatives from Penicillium polonicum.

Five new α-pyrone derivatives, named penpolonin A-E (1-5), together with two known compounds (6-7) were acquired from the endophytic fungus Penicillium polonicum isolated from the roots of Camptotheca acuminata Decne. Their structures were established by combination of NMR and HRESIMS data and the absolute configurations of 1-5 were determined by NMR calculations and comparison of experimental and calculated ECD data. Compounds 3 and 7 exhibited moderate cytotoxicity against Hep-2, TU212 human laryngeal cancer cells with IC50 values ranging from 31.6 to 45.1 µg/ml, compound 4 showed weak cytotoxicity against the Hep-2 and TU212 cell lines with IC50 values of 69.2 and 68.7 µg/ml.

Biotechnology of camptothecin production in Nothapodytes nimmoniana, Ophiorrhiza sp. and Camptotheca acuminata.

Cancer is probably the deadliest human disease in recent years. In the past few years, rapid clinical progress has been made in the field of anticancer drug development. Plant secondary metabolites have been noted as extremely efficacious as promising natural source for anticancer therapy for many years. Camptothecin (CPT) is one of the popularly used anti-tumor drugs possessing clinically proven properties against a plethora of human malignancies that include ovarian and colorectal cancers. For the first time, CPT was obtained from the extracts of a Chinese medicinal tree, Camptotheca acuminata Decne. from the family Cornaceae. Subsequently, CPT was also isolated from the bark of Nothapodytes foetida (Wight) Sleumer (Icacinaceae). However, the availability of enough natural sources for obtaining CPT is a major constraint. Due to overexploitation and harvesting, loss of habitat, excessive trading, and unfavorable environmental factors, the natural source of CPT has become extinct or extremely limited and hence they are red listed under endangered species. Conventional propagation has also failed to meet the ever-expanding demand for CPT production. With this, biotechnological toolkits have constantly been used as a boon to produce sustainable source, utilization, and ex situ conservation of medicinal plants. The approaches serve as a supplement to traditional agriculture in the mass production of plant metabolites with potent bioactivities. Non-availability of enough anticancer medicine and the requirement to satisfy current demands need a sustainable source of CPT. With this background, we present a comprehensive review on CPT discovery, its occurrence in the plant kingdom, biosynthesis, phytochemistry, pharmacological properties, clinical studies, patterns of CPT accumulation, and biotechnological aspects of CPT production in three plants, viz., N. nimmoniana, Ophiorrhiza species, and C. acuminata.Key points• Biotechnological approaches on production of camptothecin from Nothapodytes nimmoniana, Ophiorrhiza species, and Camptotheca acuminata• In vitro propagation of camptothecin-producing plants• Genetic diversity and transgenic research on camptothecin-producing plants.

Two New Nonenolides from Diaporthe sp. SXZ-19, an Endophytic Fungus of Camptotheca Acuminata.

Two new nonenolides named diaportheolides A (1) and B (2) were isolated from the endophytic fungus Diaporthe sp. SXZ-19 of Camptotheca acuminata. The chemical structures of 1 and 2 were elucidated by spectroscopic analyses, including 1D- and 2D-NMR experiments and HR-ESI-MS data analysis. Their in vitro antibacterial activities are established to be insignificant.

Hyaluronic Acid-Coated Camptothecin Nanocrystals for Targeted Drug Delivery to Enhance Anticancer Efficacy.

Tumor-targeted drug delivery via chemotherapy is very effective on cancer treatment. For potential anticancer agent such as Camptothecin (CPT), high chemotherapeutic efficacy and accurate tumor targeting are equally crucial. Inspired by special CD44 binding capability from hyaluronic acid (HA), in this study, novel HA-coated CPT nanocrystals were successfully prepared by an antisolvent precipitation method for tumor-targeted delivery of hydrophobic drug CPT. These HA-coated CPT nanocrystals demonstrated high drug loading efficiency, improved aqueous dispersion, prolonged circulation, and enhanced stability resulting from their nanoscaled sizes and hydrophilic HA layer. Moreover, as compared to crude CPT and naked CPT nanocrystals, HA-coated CPT nanocrystals displayed dramatically enhanced in vitro anticancer activity, apoptosis-inducing potency against CD44 overexpressed cancer cells, and lower toxic effect toward normal cells due to pH-responsive drug release behavior and specific HA-CD44 mediated endocytosis. Additionally, HA-coated CPT nanocrystals performed fairly better antimigration activity and biocompatibility. The possible molecular mechanism regarding this novel drug formulation might be linked to intrinsic mitochondria-mediated apoptosis by an increase of Bax to Bcl-2 ratio and upregulation of P53. Consequently, HA-coated CPT nanocrystals are expected to be an effective nanoplatform in drug delivery for cancer therapy.

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism.

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides.

Metabolite Diversity in Alkaloid Biosynthesis: A Multilane (Diastereomer) Highway for Camptothecin Synthesis in Camptotheca acuminata.

Camptothecin is a monoterpene indole alkaloid (MIA) used to produce semisynthetic antitumor drugs. We investigated camptothecin synthesis in Camptotheca acuminata by combining transcriptome and expression data with reverse genetics, biochemistry, and metabolite profiling. RNAi silencing of enzymes required for the indole and seco-iridoid (monoterpene) components identified transcriptional crosstalk coordinating their synthesis in roots. Metabolite profiling and labeling studies of wild-type and RNAi lines identified plausible intermediates for missing pathway steps and demonstrated nearly all camptothecin pathway intermediates are present as multiple isomers. Unlike previously characterized MIA-producing plants, C. acuminata does not synthesize 3-α(S)-strictosidine as its central MIA intermediate and instead uses an alternative seco-iridoid pathway that produces multiple isomers of strictosidinic acid. NMR analysis demonstrated that the two major strictosidinic acid isomers are (R) and (S) diastereomers at their glucosylated C21 positions. The presence of multiple diastereomers throughout the pathway is consistent with their use in synthesis before finally being resolved to a single camptothecin isomer after deglucosylation, much as a multilane highway allows parallel tracks to converge at a common destination. A model "diastereomer" pathway for camptothecin biosynthesis in C. acuminata is proposed that fundamentally differs from previously studied MIA pathways.

Bicarbonate stimulates non-structural carbohydrate pools of Camptotheca acuminata.

The role of root-derived dissolved inorganic carbon (DIC) has been emphasized lately, as it can provide an alternative source of carbon for photosynthesis. The fate of newly fixed DIC and its effect on non-structural carbohydrate (NSC) pools has not been thoroughly elucidated to date. To this end, we used 13 C (NaHCO3 ) as a substrate tracer to investigate the incorporation of newly fixed bicarbonate into the plant organs and NSC compounds of Camptotheca acuminata seedlings for 24 and 72 h. NSC levels across the organs were all markedly increased within 24 h of labeling treatment and afterward only decreased in stems at 72 h. The variation range of NSC concentrations in roots was considerably smaller than in the stem and leaves. As time passed, the δ13 C in NSC compounds was significantly affected by 13 C labeling and was more positive in the roots than in the stem and leaves. Starch was more 13 C-enriched than was soluble carbohydrate, and the δ13 C of root starch was as high as -4.70‰. Bicarbonate incorporation into newly formed NSC compounds contributed up to 0.24% of the root starch within 72 h. These data provided strong evidence that bicarbonate not only acted as a C source that contributed slightly to the NSC pools but also stimulated the increase in NSC pools. The present study expands our understanding of the rapid change of NSC pools across the organs in response to bicarbonate.

Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS.

Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in Camptotheca acuminata, the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the present study, a 70% aqueous acetonitrile was verified as an optimal extraction solvent for microwave-assisted extraction (MAE) of metabolites by spiking experiments. Based on multi-objective optimization, the best extraction conditions of a solid-liquid ratio of 1:20, microwave power of 230 W, and a time of 4 min were achieved using a full factorial 34 experimental design. Crude extracts obtained from the shoot apex of C. acuminata using MAE have been qualitatively profiled by high-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS/MS) and a HPLC triple quadrupole-MS (HPLC-TSQ-MS) analysis was conducted for their metabolite content in different tissues. CPT, and ten related metabolites and their isomers, including tryptamine, loganic acid, secologanic acid, strictosidinic acid, strictosamide, strictosamide epoxide, strictosamide diol, strictosamide ketolactam, pumiloside, and deoxypumiloside, were detected and tentatively identified. Scanning electron microscopy (SEM) imaging of the shoot apex demonstrated that severe cell disruption was evident after intensified extraction processes. The study showed the difference of metabolite profiles and the enhancement of metabolite content after microwave-pretreated techniques, and the established MAE procedure is an effective methodology to preserve valuable metabolite compounds for analysis.

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.

Simultaneous separation and analysis of camptothecin alkaloids in real samples by large-volume sample stacking in capillary electrophoresis.

Large-volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether-ß-cyclodextrin and 20 mm ionic liquid 1-ethyl-3-methyllimidazole l-lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 µg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.

Root-derived bicarbonate assimilation in response to variable water deficit in Camptotheca acuminate seedlings.

Water deficit is one of the key factors that limits the carbon (C) assimilation and productivity of plants. The effect of variable water deficit on recently root-derived bicarbonate assimilation in Camptotheca acuminate seedlings was investigated. Three-month-old seedlings were subjected to three water regimes, well-watered (WW), moderate stress (MS), and severe stress (SS) induced by polyethyleneglycol, in conjunction with relatively high (H) and low (L) natural 13C-abundance of NaHCO3-labeled treatments in hydroponics for 14 days. The δ13C of the newly expanded leaves in H were generally more enriched in heavy isotopes than were those in L, indicative of the involvement of bicarbonate in aboveground tissues. The C isotope fractionation of newly expanded leaves relative to air (∆13Cair-leaves) ranged from 17.78 to 21.78‰ among the treatments. The ∆13Cair-leaves under the MS and SS treatments in H were both more negative than was that in L. A linear regression between Ci/Ca and ∆13Cair-leaves in both L and H were different from the theoretical regression. On the basis of the two end-member mixing model, the proportion of fixed CO2 supplied from bicarbonate contributing to the total photosynthetically inorganic C assimilation were 10.34, 20.05 and 16.60% under the WW, MS, and SS treatments, respectively. These results indicated that the increase in water deficit decreased the atmospheric CO2 gain but triggered a compensatory use of bicarbonate in C. acuminate seedlings.