The vacuolar localization of a basic peroxidase isoenzyme responsible for the synthesis of α-31,41-anhydrovinblastine in Catharanthus roseus (L.) G. Don Leaves

Partially purified protein extracts of Catharanthus roseus leaves were able to couple catharanthine and vindoline to produce α-3′,4′-anhydrovinblastine (AVLB) in a reaction strictly dependent on H₂O₂. This result, and the co-purification of peroxidase with AVLB synthetase activity, strongly suggest a peroxidase-like nature for the coupling enzyme. Only one peroxidase isoenzyme was detected in C. roseus leaves, and it was shown that this isoenzyme consists of a molecularly-heterogeneous basic peroxidase (EC 1-11-1-7) mainly located in the vacuole. These results suggest that a basic peroxidase located in the vacuole may be the main enzyme responsible for AVLB synthesis in C. roseus leaves. This isoenzyme was also found in cell walls where a peroxidase inhibitor was detected.     

长春花(Catharanthus roseus)中吲哚类生物碱含量的比较

建立了反相高效液相法测定长春花中吲哚类生物碱文多灵、长春质碱和阿玛碱含量的方法,色谱柱为HiQ sil C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为1%二乙胺水溶液(磷酸调pH=7.2)—甲醇—乙腈/2:1:1(V/V);流速为1 mL·min^-1;检测波长为215 nm;柱温为40℃。并采用此方法对长春花根、茎、叶、花和种子以及不同产地的长春花中的这3种生物碱进行了检测,结果表明文多灵和长春质碱主要存在于在根、茎、叶、花中,阿玛碱主要存在于种子中;并且随着地理位置的北移,长春花中的文多灵和长春质碱的含量逐渐降低,温室中人为控制长春花中的栽培条件能提高二者的含量。     

Annuario della Società Botanica Italiana 1932

A sandy culture experiment was conducted to investigate the effects of exogenous CaCl₂ on the indole alkaloid accumulation in Catharanthus roseus under salt stress. One-month seedlings of C. roseus were treated with the different concentrations of NaCl (0, 50, and 100 mmol l^? 1) and 7.5 mmol l^? 1 CaCl₂. The plant samples were analyzed after 7 days of the treatments. The NaCl-stressed plants showed decrease of fresh and dry weight and increase of malondialdehyde (MDA) content compared to control. Tryptophan decarboxylase (TDC) activity increased significantly under 50 mmol l^? 1 NaCl without CaCl₂ addition, 50 mmol l^? 1 NaCl with 7.5 mmol l^? 1 CaCl₂, and CaCl₂ treatment without NaCl addition. There was a significant increase in peroxidase activity under NaCl stress compared to control. The vindoline, catharanthine, vincristine, and vinblastine contents increased under salt stress (especially with 50 mmol l^? 1 NaCl treatment with or without CaCl₂). Addition of CaCl₂ to NaCl-stressed plants increased biomass, TDC activity, vindoline, and catharanthine contents and lowered MDA and vincirstine contents compared to the plants without CaCl₂. The plants treated with CaCl₂ alone showed higher TDC activity, vindoline, catharanthine, and vinblastine content when compared to control. The results showed that exogenous CaCl₂ could promote the indole alkaloid metabolism under salt stress.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

浙江富阳和海南文昌人工种植长春花生物碱积累组织特异性对比

药用植物长春花的人工栽培分布区主要在我国海南省,目前已引种扩大到浙江、四川等地。为了研究不同栽培区长春花生物碱积累的区域性差异和组织特异性分布特点,本文以浙江富阳和海南文昌两地所产的同苗龄长春花为原料,对其不同部位的长春碱、文朵灵和长春质碱的含量进行对比,分析3种生物碱在不同部位中积累的相关性。研究结果表明,海南文昌所产长春花植株的叶片中3种生物碱含量均高于浙江富阳。但分枝中长春碱含量特点是浙江富阳要显著高于海南文昌;不同部位生物碱之间相关性分析表明,叶片和主茎中文朵灵和长春质碱含量呈显著正相关,而分枝中长春碱与其前体长春质碱成极显著负相关。本文为长春花人工栽培区分布评估提供了理论指导。     

Mechanism of interaction of vinca alkaloids with tubulin: catharanthine and vindoline

The interactions of the vinca alkaloid drugs catharanthine and vindoline with tubulin have been investigated and compared with those of vinblastine and vincristine. Both drugs were found to be less effective in bringing about the inhibition of tubulin self-assembly into microtubules than vincristine and vinblastine, the drug to protein molar ratio required being 3 orders of magnitude greater. An analytical ultracentrifuge study has shown that catharanthine can induce the self-association of tubulin into linear indefinite polymers with an efficacy that is 75% that of vinblastine or vincristine, the intrinsic dimerization constant for the liganded protein being K2 congruent to 1 x 10(5) M-1. The effect of vindoline was marginally detectable. Binding studies of catharanthine using the gel batch and fluorescence perturbation techniques showed a polymerization-linked binding of one catharanthine molecule per tubulin alpha-beta dimer with a binding constant of (2.8 +/- 0.4) x 10(3) M-1. For vindoline, binding to tubulin was marginally detectable by fluorescence spectroscopy, although addition of vindoline to tubulin did generate a difference spectrum. It was concluded that the binding of vinblastine/vincristine to tubulin and its consequences are determined by the interaction of the indole part of catharanthine with tubulin, the role of vindoline being that of an anchor.     

Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P-450 dependent monooxygenase.

An end-product indole alkaloid, catharanthine, inhibits a membrane-bound cytochrome P-450 dependent monooxygenase of the higher plant, $\text{Vinca rosea}$. Kinetic analysis revealed the alkaloid to be a reversible, linear, non-competitive inhibitor (K_i=1 mM) with respect to its substrates, geraniol and NADPH. Comparable inhibition of the solubilized monooxygenase by catharanthine tends to exclude a mechanism based upon disruption of membrane organization. On the basis of its inhibition of solubilized hydroxylase in the presence and absence of sodium cholate, it is also unlikely that catharanthine competes for putative phospholipid binding site(s). Two additional end-product alkaloids, vinblastine and vindoline were less inhibitory. Since the hydroxylase catalyzes one of the first committed steps in the biosynthesis of indole alkaloids, these observations suggest feedback control of the pathway by catharanthine.     

人工种植长春花生物学性状和生物碱含量的季节动态

以人工种植的长春花（Catharanthus roseus（L.）G. Don）为材料,研究了一个生长季节内长春花的生物学性状和生物碱含量的季节动态。研究发现,长春花的株高、生物量和叶片数呈现相似的生长趋势,即前期（5~7月）缓慢增长、中期（7~9月）快速增长和后期（9~10月）增长缓慢3个明显的季节生长特征,而花总数在前期和中期也呈现相似的规律,但在后期（9~10月）快速下降。长春花叶片中3 种生物碱含量的季节变化规律明显,变化趋势相一致。3 种生物碱的含量在一个生长季节内均是先增加后降低再增加的趋势,且3 种生物碱含量均在7月下旬和10月下旬出现2个明显的高峰值。长春花叶片中长春碱含量与长春质碱和文多灵含量之间均有较强的正相关,且与文多灵含量呈显著的正相关（p<0.05）,长春花叶片中长春质碱含量与文多灵含量之间呈显著的正相关（p<0.05）。因此,生产实践中人工种植长春花的最佳采收期是10月末,并可通过改变环境因子进一步诱导长春花叶片中长春碱类物质的积累。     

Traditional and non-traditional plant growth regulators alters phytochemical constituents in Catharanthus roseus

The effect of different plant growth regulators (PGR) and elicitor treatments on the alkaloid profile variation of Catharanthus roseus was investigated in the present study. The PGR used were paclobutrazol (PBZ), gibberellic acid (GA₃) and Pseudomonas fluorescens elicitors (PF Elicitors). The estimated alkaloids were ajmalicine, catharanthine, tabersonine, serpentine and vindoline. In roots, the ajmalicine content increased significantly under all the treatments on all sampling days. In roots, the catharanthine contents increased with the age in control and growth regulator treatments, but the increase was not prominent and significant in PGR treatments when compared to controls. The serpentine contents of the plant increased with PGR treatments, but the increase was more prominent in PBZ treatments when compared to other treatments. The increase was in the order PBZ > PF Elicitors > GA₃. C. roseus never showed any significant increase in tabersonine contents in the roots under GA₃ treatments, but it increased significantly under PBZ and PF Elicitors when compared to control plants. The root vindoline contents increased with PBZ and PF Elicitors treatments but the decreased under GA₃ treatments when compared to control plants. Our results have good significance, as these increases the secondary metabolites of this traditional medicinal plant.     

Effects of elevated CO2 and increased nitrogen deposition on photosynthesis and growth of understory plants in spruce model ecosystems

We studied the effects of atmospheric CO₂ enrichment (280, 420 and 560 l CO₂ l^–1) and increased N deposition (0,30 and 90 kg ha^–1 year^–1) on the spruce-forest understory species Oxalis acetosella, Homogyne alpina and Rubus hirtus. Clones of these species formed the ground cover in nine 0.7 m² model ecosystems with 5-year-old Picea abies trees (leaf area index of approx 2.2). Communities grew on natural forest soil in a simulated montane climate. Independently of N deposition, the rate of light-saturated net photosynthesis of leaves grown and measured at 420 l CO₂ l^–1 was higher in Oxalis and in Homogyne, but was not significantly different in Rubus compared to leaves grown and measured at the pre-industrial CO₂ concentration of 280 l l^–1. Remarkably, further CO₂ enrichment to 560 l l^–1 caused no additional increase of CO₂ uptake. With increasing CO₂ supply concentrations of non-structural carbohydrates in leaves increased and N concentrations decreased in all species, whereas N deposition had no significant effect on these traits. Above-ground biomass and leaf area production were not significantly affected by elevated CO₂ in the more vigorously growing species O. acetosella and R. hirtus, but the slow growing H. alpina produced almost twice as much biomass and 50% more leaf area per plant under 420 l CO₂ l^–1 compared to 280 l l^–1 (again no further stimulation at 560 l l^–1). In contrast, increased N addition stimulated growth in Oxalis and Rubus but had no effect on Homogyne. In Oxalis (only) biomass per plant was positively correlated with microhabitat quantum flux density at low CO₂, but not at high CO₂ indicating carbon saturation. On the other hand, the less shade-tolerant Homogyne profited from CO₂ enrichment at all understory light levels facilitating its spread into more shady micro-habitats under elevated CO₂. These species-specific responses to CO₂ and N deposition will affect community structure. The non-linear responses to elevated CO₂ of several of the traits studied here suggest that the largest responses to rising atmospheric CO₂ are under way now or have already occurred and possible future responses to further increases in CO₂ concentration are likely to be much smaller in these understory species.     

Biomimetic one-pot synthesis of vinblastine: NAD(P)H-mediated vinblastine synthesis from the product of FMN-mediated vindoline–catharanthine coupling under near-ultraviolet light

Vinblastine (VLB) is an antineoplastic agent contained in leaves of Catharanthus roseus. One-pot synthesis of VLB from vindoline and catharanthine, biosynthetic precursors in the plant, was achieved under biomimetic conditions. FMN-mediated coupling of vindoline and catharanthine under irradiation with near-ultraviolet light was followed by NAD(P)H-mediated direct conversion of the coupling product to VLB in the dark. The yield of VLB was governed by the level of NAD(P)H added, and the highest yield was obtained by incubation with 10 mM NAD(P)H for 5 h. It is postulated, as one concept of VLB biosynthesis, that similar non-enzymic VLB synthesis may occur in the plant leaves.     

Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus

The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

High-light effects on CO2 fixation gradients across leaves

Chlorophyll fluorescence and internal patterns of ¹⁴CO₂ fixation were measured in sun and shade leaves of spinach after treatment with various light intensities. When sun leaves were irradiated with 2000μmol m^?2 s^?1 for 2h, F_V/F_M decreased by about 15%, but ¹⁴CO₂ fixation was unaffected, whereas shade leaves exhibited a 21% decrease in F_v/F_M and a 25% decrease in ¹⁴CO₂ fixation. Irradiation of sun and shade leaves with 4000μmol m^?1 for 4 h decreased F_V/F_M by 30% in sun leaves and 40% in shade leaves, while total ¹⁴CO₂ fixation decreased by 41% in sun leaves and 55% in shade leaves. After light treatment, gradients of CO₂ fixation across leaves were determined by measuring ¹⁴CO₂ fixed in paradermal leaf sections after a 10s pulse of ¹⁴CO₂. Gradients of ¹⁴CO₂ fixation in control sun and shade leaves were identified when expressed on a relative basis and normalized for leaf depth. Treatment of leaves with 2000 μmol PAR m^?2 s^?1 for 2h did not after patterns of carbon fixation across sun leaves, but slightly altered the pattern in shade leaves. In contrast, treatment of sun and shade leaves with 4000μmol m^?2 s^?1 for 4h decreased carbon fixation more in the palisade mesophyll cells than in the spongy mesophyll cells of sun and shade leaves, and fixation in medial tissue of shade leaves was dramatically decreased compared to the adaxial and abaxial tissue. The interaction between leaf anatomy and biochemical parameters involved in tolerance to photoinhibition in spinach is discussed.     

Developmental Change in CO2 Compensation Concentrations in Spartina alterniflora Results from Sigmoidal Photosynthetic CO2 Responses

We investigated seasonal patterns of photosynthetic responses to CO₂ concentrations in Spartina alterniflora Loisel, an aerenchymous halophyte grass, from a salt marsh of the Bay of Fundy (NB, Canada), and from plants grown from rhizome in controlled-environment chambers. From late May to August, CO₂ compensation concentrations () of field-grown leaves varied between 2.5–10.7 cm³(CO₂) m^–3, with a mean of 5.4 cm³(CO₂) m^–3. From September onwards field leaves showed CO₂ compensation concentrations from 6.6–21.1 cm³(CO₂) m^–3, with a mean of 13.1 cm³ m^–3 well into the C₃–C₄ intermediate range. The seasonal variability in did not result from changing respiration, but rather from a sigmoidal response of net photosynthetic rate (P _N) to applied CO₂ concentration, found in all tested leaves but which became more pronounced late in the season. One explanation for the sigmoidal response of P _N to external CO₂ concentration could be internal delivery of CO₂ from roots and rhizomes to bundle sheath cells via the aerenchyma, but the sigmoidal responses in S. alterniflora persisted out to the tips of leaves, while the aerenchyma extend only to mid-leaf. The sigmoidicity persisted when CO₂ response curves were measured from low to high CO₂, or from high to low CO₂, and even when prolonged acclimation times were used at each CO₂ concentration.