Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Studies on leaf spot disease of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its impact on secondary metabolites

During an investigation of the disease profile of Withania somnifera, it was observed that leaf spot is the most prevalent disease. Repeated isolations from infected leaf tissues and pathogenicity tests showed the association of fungal pathogen identified as Alternaria alternata (Fr.) Keissler. Scanning electron microscopy showed various histological changes in the leaf tissues of infected plants. A decrease in total content of reducing sugars (20%) and chlorophyll (26.5%) was observed in diseased leaves whereas an increase was noticed in proline (25%), free amino acids (3%) and proteins (74.3%). High performance thin layer chromatography (HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and withanolides contents by 15.4% and 76.3% respectively, in contrast to an increase in total alkaloids by 49.3%, information hitherto unreported in W. somnifera.     

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.     

New insights in phytoplasma‐vector interaction: acquisition and inoculation of flavescence dorée phytoplasma by Scaphoideus titanus adults in a short window of time

The leafhopper Scaphoideus titanus is able to transmit 16SrV phytoplasmas agents of grapevine's flavescence dorée (FD) within 30–45 days, following an acquisition access period (AAP) of a few days feeding on infected plants as a nymph, a latency period (LP) of 3–5 weeks becoming meanwhile an adult, and an inoculation access period (IAP) of a few days on healthy plants. However, several aspects of FD epidemiology suggest how the whole transmission process may take less time, and may start directly with adults of the insect vector. Transmission experiments have been set up under lab condition. Phytoplasma‐free S. titanus adults were placed on broad bean (BB) plants (Vicia faba) infected by FD‐C (16SrV‐C) phytoplasmas for an AAP = 7 days. Afterwards, they were immediately moved onto healthy BB for IAP, which were changed every 7 days, obtaining three timings of inoculation: IAP 1, IAP 2 and IAP 3, lasting 7, 14 and 21 days from the end of AAP, respectively. DNA was extracted from plants and insects, and PCR tests were performed to identify FD phytoplasmas. Insects were dissected and fluorescence in situ hybridisation was made to detect the presence of phytoplasmas in midguts and salivary glands. The rate of infection in insects ranged 46–68% without significant differences among IAPs. Inoculation in plants succeeded in all IAPs, at a rate of 16–23% (no significant differences). Phytoplasma load was significantly higher in IAP 3 than IAP 1–2 for both plants and insects. Phytoplasmas were identified both in midgut and salivary glands of S. titanus at all IAP times. The possible implications of these results in the epidemiology of flavescence dorée are discussed.     

Occurrence of Phytoplasmas Related to Stolbur and to ‘Candidatus Phytoplasma japonicum’ in Woody Host Plants in China

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to ‘Candidatus Phytoplasma japonicum’. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.     

Histopathology and polyphenol content in plants infected by phytoplasmas

The alterations of cell walls and the localization of several compounds such as polyphenols, suberin, lignin, in plum and apple plants infected with plum leptonecrosis (PLN) and apple proliferation (AP) phytoplasmas respectively, were investigated. Catharanthus roseus plants, infected with AP or PLN were also studied. The 4,6-diamidino-2-phenylindole (DAPI) test and transmission electron microscopy showed the presence of phytoplasmas in all infected plants. Specific histological stainings for cutinized/suberinized cell walls, tannin deposits and vacuolar polyphenol inclusions, performed on leaf and stem tissues, revealed an increase of these substances in infected plum and apple plants. No differences occurred in C. roseus. Total polyphenol analysis confirmed a strong increase (3-fold) in the polyphenol content in infected tissues, particularly in plum leaves. From the data obtained it appears that polyphenols can be considered as defence-related metabolites in plum and apple plants infected by phytoplasmas. Further investigations are necessary to determine whether these compounds play a specific role in the development of all phytoplasma/host interactions and in the defence-related processes.     

Effects of ‘Candidatus Phytoplasma asteris’ on the Volatile Chemical Content and Composition of Grindelia robusta Nutt.

Grindelia robusta, a perennial herb, contains an essential oil that is used as an antitussive, sedative, and analgesic agent. During the spring of 2007, ‘Candidatus Phytoplasma asteris’‐related phytoplasmas were identified in plants showing virescence and phyllody symptoms. The qualitative and quantitative composition of the oil of healthy and infected plants was compared by gas chromatography/mass spectrometry. Samples from six symptomatic and five asymptomatic plants tested by nested PCR followed by RFLP analyses confirmed the presence of ‘Ca. P. asteris’ in all symptomatic samples. The oils from healthy and infected plants, obtained by steam distillation, contained 42 components; that of healthy plants contained a higher concentration of monoterpenes, especially limonene and bornyl acetate, which were nearly 50% higher.     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

Mixed Infection of Two Groups (16SrI & V) of Phytoplasmas in a Single Jujube Tree in China

Symptoms similar to Jujube witches' broom disease were observed on jujube (Zizyphus jujuba) plants in an orchard in Xuchang City, Henan Province, China. Phytoplasmas were detected in one sample from symptomatic plants by nested PCR assay employing 16S rRNA gene primers P1/P7 followed by R16F2n/R16R2. Virtual RFLP analysis of the resulting products (F2nR2 region) shown that total of two different groups (16SrI and 16SrV) phytoplasmas associated with the infected jujube. This is the first report of phytoplasmas mixed infection of jujube in China.     

Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae)

Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.     

Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina

Two phytoplasmas closely related to the X‐disease group were associated with China‐tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China‐tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China‐tree and garlic decline phytoplasmas to the 16SrIII (X‐disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China‐tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X‐disease group.     

Seasonal Colonization Pattern of European Stone Fruit Yellows Phytoplasmas in Different Prunus Species Detected by Specific PCR

The seasonal variation in the colonization of two Japanese plum trees by European stone fruit yellows (ESFY) phytoplasmas was followed by polymerase chain reaction (PCR) detection for 2 years. Samples were obtained monthly from nine above-ground sampling sites and one root. The colonization of the trees was systemic from July until leaf fall. The ESFY phytoplasmas were also detected in off-season grown leaves during winter until March. In contrast, almost no phytoplasmas could be detected in normally grown leaves in April and May. Similar results have been obtained for European plum cultivars using 4′,6-diamidino-2-phenylindole (DAPI) staining and PCR and for apricot cultivars tested by PCR. A possible explanation of this phenomenon is discussed. The root system of the corresponding Prunus marianna GF 8–1 rootstocks remained infected throughout the year and the phytoplasmas were equally distributed within the roots as determined when the trees were uprooted. In vitro culture was used to demonstrate that ESFY phytoplasmas detected by PCR in winter in aerial parts of the tree were viable. Nine ESFY-diseased shoot cultures were obtained at four different time points during winter. Sampling protocols for ESFY phytoplasma detection by PCR in routine diagnosis are discussed.     

Euscelis incisus (Cicadellidae,Deltocephalinae), a natural vector of 16SrIII‐B phytoplasma causing multiple inflorescence disease of Cirsium arvense

We investigated multiple inflorescence disease of Cirsium arvense (CMI) and its association with phytoplasmas of the 16SrIII‐B subgroup, potential natural vector(s) and reservoir plant(s). From five locations in northern Serbia, 27 plants of C. arvense, 1 C. vulgare and 3 Carduus acanthoides with symptoms of multiple inflorescences (MIs) were collected and tested for 16SrIII group phytoplasmas. All symptomatic plants were found to be infected. Tentative reservoir plants and insect vectors were collected at a Dobanovci site where the continuous presence of CMI disease was recorded. Among the 19 most abundant plant species submitted to phytoplasma testing, all symptomless, the presence of the 16SrIII group was detected only in two legumes: Lathyrus tuberosus (2/5) and L. aphaca (1/5). Among 19 insect species from six families of Auchenorrhyncha, the deltocephalid leafhopper Euscelis incisus was the only insect carrying a 16SrIII phytoplasma (10% of analysed individuals). Transmission trials were performed with naturally infected E. incisus adults of the summer generation and with a laboratory population reared on red clover. After an acquisition period of 48 h on C. arvense symptomatic for MIs and a latent period of 28 days, 83% of the E. incisus adults (300/360) were infected with CMI phytoplasma. In two transmission tests, the leafhoppers successfully transmitted the phytoplasma to exposed plants (C. arvense and periwinkle), proving its role as a natural vector. Test plants of C. arvense infected with the 16SrIII‐B phytoplasma expressed typical symptoms similar to those observed in the field, such as MIs or the absence of flowering, shortened internodes and plant desiccation. Typical symptoms in infected periwinkles were virescence and phyllody. The molecular characterisation of the CMI phytoplasma isolates from diseased and asymptomatic field‐collected plants, vectors, and test plants was performed by sequence analyses of the 16S rRNA, rpl22‐rps3 and rpl15‐secY genes. Phylogenetic analyses of other members of the 16SrIII group of phytoplasmas indicated closest relatedness with clover yellow edge phytoplasma (CYE) of the 16SrIII‐B subgroup.     

Phytoplasma distribution in coconut palms affected by lethal yellowing disease

Lethal yellowing (LY), the most devastating disease affecting the coconut palm in America, is caused by phytoplasmas known to be distributed in different parts of infected plants. However, no comprehensive reports exist on the phytoplasma distribution. This study refers to the detection of LY phytoplasma DNA using PCR in different coconut plant parts, throughout the development of the disease. Sample analysis of positive palms taken at different stages of disease development (either symptomatic or symptomless) showed differences in the percentage of LY detection between plant parts. Some parts showed a very high level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, whereas no LY phytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless‐infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) stem, prior to the appearance of symptoms, suggest that this part of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested‐PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY‐positive symptomatic palms was 58% and from LY‐negative symptomless palms were 71%. No phytoplasma was detected in seedlings tested from both symptomatic and non‐symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.     

Transgene expression in strawberries driven by a heterologous phloem-specific promoter

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the -glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H⁺ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-M thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Influence of freezing and non-freezing temperature on somatic embryogenesis and vinblastine production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don.

Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.