Ornamental exterior versus therapeutic interior of Madagascar periwinkle (Catharanthus roseus): the two faces of a versatile herb.

Catharanthus roseus (L.) known as Madagascar periwinkle (MP) is a legendary medicinal plant mostly because of possessing two invaluable antitumor terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The plant has also high aesthetic value as an evergreen ornamental that yields prolific blooms of splendid colors. The plant possesses yet another unique characteristic as an amiable experimental host for the maintenance of the smallest bacteria found on earth, the phytoplasmas and spiroplasmas, and serves as a model for their study. Botanical information with respect to synonyms, vernacular names, cultivars, floral morphology, and reproduction adds to understanding of the plant while the geography and ecology of periwinkle illustrate the organism's ubiquity. Good agronomic practices ensure generous propagation of healthy plants that serve as a source of bioactive compounds and multitudinous horticultural applications. The correlation between genetic diversity, variants, and TIA production exists. MP is afflicted with a whole range of diseases that have to be properly managed. The ethnobotanical significance of MP is exemplified by its international usage as a traditional remedy for abundant ailments and not only for cancer. TIAs are present only in micro quantities in the plant and are highly poisonous per se rendering a challenge for researchers to increase yield and reduce toxicity.

Correspondence between flowers and leaves in terpenoid indole alkaloid metabolism of the phytoplasma-infected Catharanthus roseus plants.

Several plants of Catharanthus roseus cv 'leafless inflorescence (lli)' showing phenotype of phytoplasma infection were observed for symptoms of early flowering, virescence, phyllody, and apical clustering of branches. Symptomatic plants were studied for the presence/absence and identity of phytoplasma in flowers. Transcription levels of several genes involved in plants' metabolism and development, accumulation of pharmaceutically important terpenoid indole alkaloids in flowers and leaves and variation in the root-associated microbial flora were examined. The expression profile of 12 genes studied was semi-quantitatively similar in control leaves and phytoplasma-infected leaves and flowers, in agreement with the symptoms of virescence and phyllody in phytoplasma-infected plants. The flowers of phytoplasma-infected plants possessed the TIA profile of leaves and accumulated catharanthine, vindoline, and vincristine and vinblastine in higher concentrations than leaves. The roots of the infected plants displayed lower microbial diversity than those of normal plants. In conclusion, phytoplasma affected the biology of C. roseus lli plants multifariously, it reduced the differences between the metabolite accumulates of the leaves and flowers and restrict the microbial diversity of rhizosphere.

Phytoplasma-induced floral abnormalities in Catharanthus roseus are associated with phytoplasma accumulation and transcript repression of floral organ identity genes.

Floral symptoms caused by phytoplasma largely resemble floral reversion in other plants. Periwinkle leaf yellowing (PLY) phytoplasma and peanut witches'-broom (PnWB) phytoplasma caused different degrees of floral abnormalities on infected periwinkle plants. The PLY phytoplasma-infected plants exhibited floral discoloration, virescence, small flowers, and only occasionally full floral reversion. In contrast, PnWB phytoplasma frequently induced complete floral reversion and resulted in a witches'-broom symptom from the floral reversion. Although different degrees of floral symptoms were induced by these two phytoplasmas, the morphological disorders were similar to those of other plants carrying SEPALLATA mutations or gene silencing. Here, we compared expression levels of organ-identity-related genes and pigmentation genes during floral symptom development. Accumulation of phytoplasmas in malformed flowers and their closely surrounding leaves was also compared. In infected plants, transcript abundance of all examined organ identity genes and pigmentation genes was suppressed. Indeed, CrSEP3, a SEPALLALA3 ortholog, showed the greatest suppression among genes examined. Of the pigmentation genes, transcript reduction of chalcone synthase was most highly correlated with the loss in floral pigmentation. Floral symptom severities were associated with the accumulation of either phytoplasmas. Interestingly, both phytoplasmas accumulated to higher levels in malformed flowers than in their surrounding leaves. Many plant pathogens manipulate host plant development to their advantage. It is intriguing to see whether phytoplasmas alter floral development to increase their population.

Gene expression profiling of phytoplasma-infected Madagascar periwinkle leaves using differential display.

Phytoplasmas are small (0.2-0.8 µm), wall-less, pleiomorphic prokaryotes responsible of numerous economically important plant diseases. They are characterized by a very small genome and are obligate parasites of phloem tissues and some insects that act as vectors of infection. To investigate molecular mechanisms involved in pathogenesis, the differential display technique was here applied to identify plant genes whose transcription was significantly altered in leaves of Madagascar periwinkle (Catharanthus roseus (L.) G.Don) infected by 'Candidatus Phytoplasma pyri'. We detected, reamplified, cloned, and sequenced 16 putative differentially expressed cDNA fragments. Northern blot analysis revealed that seven of the 16 genes identified were up-regulated following phytoplasma infection, while three genes were down-regulated. The remaining six genes did not show significant changes in the level of expression. Identified genes are mainly involved in plant defence/stress responses, protein metabolism and transport, transcriptional regulation, vesicle trafficking, and carbohydrate metabolism. The possible role played by these genes in the phytoplasma infection is discussed.

[Analysis of catharanthine content and agronomic traits in Catharanthus roseus].

Catharanthine content and agronomic traits in major Catharanthus roseus varieties were analyzed. It was found that there existed great difference in catharanthine content and agronomic traits among the varieties. Catharanthine content was the highest in variety Pacifica Polka Dot (PPD), reaching 3.79 mg g(-1) dry leaf weight, and the lowest in variety Cooler Pink (CP) with only 0.9 mg g(-1) dry leaf weight. Correlation existed in certain extent between catharanthine content and agronomic traits in C. roseus. Path analysis showed that among all the agronomic traits analyzed, internodal distance positively affected catharanthine content at significant level (P<0.05), with the path coefficient being 1.473. This study provides useful information for high-catharanthine content C. roseus introduction and breeding.

Inheritance of pollen-less anthers and "thrum" and "pin" flowers in periwinkle.

Two mutants, 1 with small, pollen-less anthers (OR-EA) and another with "pin" flowers (EMS 13-2), in contrast to "thrum" flowers found in normal periwinkle (Catharanthus roseus) plants, were isolated after induced mutagenesis in strain OR and cultivar, "Dhawal," respectively. Inheritance of these 2 traits, pollen-less anthers, and pin flowers was studied by crossing the mutants with their respective parental strains. Segregation ratios observed in F(2) and testcross generations of the cross OR-EA x OR suggested that the pollen-less anthers trait was determined by duplicate recessive genes. Data obtained from F(2) and F(3) generations of the cross involving mutant EMS 13-2 with pin flowers and its parental variety Dhawal, suggested that production of pin (mutant) and thrum (normal) flowers was under the control of inhibitory epistatic interaction between 2 independently inherited genes.

Alterations in morphological parameters and photosynthetic pigment responses of Catharanthus roseus under soil water deficits.

An experiment was conducted in two varieties, rosea and alba, of Catharanthus roseus plants with two watering treatments viz., 100 and 60% of field capacity, to understand the effects of water deficit on early growth, biomass allocation and photosynthetic pigment responses. We found that there were significant differences in early growth, dry matter accumulation and pigment variations between the two varieties. The root length, shoot length, total leaf area, fresh and dry weights were significantly reduced under water stress treatments. There was a significant reduction in the photosynthetic pigment contents in both the varieties. The rosea variety was more affected due to water deficit when compared to alba variety.

Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress.

The effect of plant growth promoting rhizobacteria (PGPR) like Pseudomonas fluorescens on growth parameters and the production of ajmalicine were investigated in Catharanthus roseus under drought stress. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress and drought stress with Pseudomonas fluorescens at 1mgl(-1) and 1mgl(-1)Pseudomonas fluorescens alone from 30 days after planting (DAP) and regular irrigation was kept as control. The plants were uprooted on 41 DAS (10 DID), 46 DAS (15 DID) and 51 DAS (20 DID). Drought stress decreased the growth parameters and increased the ajmalicine content. But the treatment with Pseudomonas fluorescens enhanced the growth parameters under drought stress and partially ameliorated the drought induced growth inhibition by increasing the fresh and dry weights significantly. The ajmalicine content was again increased due to Pseudomonas fluorescens treatment to the drought stressed plants. From the results of this investigation, it can be concluded that, the seedling treatments of native PGPRs can be used as a good tool in the enhancement of biomass yield and alkaloid contents in medicinal plants, as it provides an eco-friendly approach and can be used as an agent in water deficit stress amelioration.

Construction of genetic linkage map of the medicinal and ornamental plant Catharanthus roseus.

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F(2) population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs.

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid-monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid-monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.

Allelic differences at two loci govern different mechanisms of intraflower self-pollination in self-pollinating strains of periwinkle.

Periwinkle [Catharanthus roseus (L.) G. Don], an ornamental and medicinal plant, is a self-compatible, insect-pollinated plant species in which intraflower self-pollination does not occur because of spatial separation of the stigma and anthers. Recently three self-pollinating strains-MJ, VI, and OR-were identified. Self-pollination in these strains was found to be brought about by continuous increase in gynoecium length from anthesis to self-pollination, in contrast to non-self-pollinating strains, in which the stigma remained below the base of the anthers from anthesis to flower drop. Self-pollination in these strains was found to be controlled by duplicate, recessive genes. Self-pollination in strains MJ and VI was brought about by an increase in gynoecium length resulting from an increase in the length of the ovary, while in the strain OR, the increase in gynoecium length was because of an increase in the length of the style from anthesis to self-pollination. The three strains were intercrossed to determine the relationship between genes governing self-pollination in these strains. The F(1) plants and all plants of the F(2) generation of the cross MJ x VI exhibited self-pollination that was brought about by an increase in the length of the ovary, indicating that the same genes were involved in these two strains. The F(1) plants of crosses OR x MJ and OR x VI, exhibited self-pollination that was brought about by an increase in the length of the ovary, indicating that self-pollination brought about by an increase in the length of the ovary was dominant over self-pollination brought about by an increase in the length of the style. In the F(2) and backcross [(OR x MJ) x OR and (OR x VI) x OR] generations, both self-pollinating and non-self-pollinating plants were observed. The ratio of plants with self-pollination brought about by an increase in the length of the ovary, non-self-pollinating plants, and plants with self-pollination brought about by an increase in the length of the style in the F(2) and backcross generations fit 9:6:1 and 1:2:1 ratios, respectively. All plants of the backcrosses [(OR x MJ) x MJ and (OR x VI) x VI] exhibited self-pollination brought about by an increase in the length of the ovary. The results thus supported the earlier finding that self-pollination in the studied strains was controlled by duplicate, recessive genes and suggested that three alleles at two loci determine the occurrence or nonoccurrence of intraflower self-pollination in periwinkle.