Generous hosts: Growth of ‘Candidatus Liberibacter asiaticus’ in Madagascar periwinkle (Catharanthus roseus) highlights its nutritional needs

‛Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening is not available in pure culture yet. In addition to trees of citrus and citrus relatives, ‛Ca. L. asiaticus’ can grow in Madagascar periwinkle (Catharanthus roseus). Using GC-MS, we compared the phloem sap composition in sweet orange ‛Valencia’ (Citrus sinensis) and periwinkle plants after the infection with ‛Ca. L. asiaticus’. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared to uninfected phloem sap, we found that the organic acids implicated in the TCA cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced the translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids likely benefits the bacterial growth and may explain the unequal distribution of ‘Ca L. asiaticus’ within infected trees. These findings may help in designing media for the pure culturing of ‛Ca. L. asiaticus’.

Terpenoid indole alkaloid biosynthesis in Catharanthus roseus: effects and prospects of environmental factors in metabolic engineering

Plants synthesize a vast array of specialized metabolites that primarily contribute to their defense and survival under adverse conditions. Many of the specialized metabolites have therapeutic values as drugs. Biosynthesis of specialized metabolites is affected by environmental factors including light, temperature, drought, salinity, and nutrients, as well as pathogens and insects. These environmental factors trigger a myriad of changes in gene expression at the transcriptional and posttranscriptional levels. The dynamic changes in gene expression are mediated by several regulatory proteins that perceive and transduce the signals, leading to up- or down-regulation of the metabolic pathways. Exploring the environmental effects and related signal cascades is a strategy in metabolic engineering to produce valuable specialized metabolites. However, mechanistic studies on environmental factors affecting specialized metabolism are limited. The medicinal plant Catharanthus roseus (Madagascar periwinkle) is an important source of bioactive terpenoid indole alkaloids (TIAs), including the anticancer therapeutics vinblastine and vincristine. The emerging picture shows that various environmental factors significantly alter TIA accumulation by affecting the expression of regulatory and enzyme-encoding genes in the pathway. Compared to our understanding of the TIA pathway in response to the phytohormone jasmonate, the impacts of environmental factors on TIA biosynthesis are insufficiently studied and discussed. This review thus focuses on these aspects and discusses possible strategies for metabolic engineering of TIA biosynthesis. Purpose of work Catharanthus roseus is a rich source of bioactive terpenoid indole alkaloids (TIAs). The objective of this work is to present a comprehensive account of the influence of various biotic and abiotic factors on TIA biosynthesis and to discuss possible strategies to enhance TIA production through metabolic engineering.

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

Response of Common Garden Annuals to Sublethal Rates of 2,4-D and Dicamba with or without Glyphosate

An experiment was conducted in 2017 and 2018 to determine the sensitivity of common garden annuals to sublethal rates of 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba with or without glyphosate. Sublethal rates corresponding to 1/10×, 1/100×, and 1/300× of the full labeled rate (1×) of 2,4-D (1.0 lb/acre), 2,4-D plus glyphosate (1.0 lb/acre plus 1.0 lb/acre), dicamba (0.5 lb/acre), and dicamba plus glyphosate (0.5 lb/acre plus 1.0 lb/acre) were applied to ‘Prelude’ wax begonia (Begonia ×semperflorens-cultorum), ‘Wizard’ coleus (Solenostemon scutellarioides), ‘Pinto’ zonal geranium (Pelargonium ×hortorum), ‘Dazzler’ impatiens (Impatiens walleriana), ‘Bonanza’ french marigold (Tagetes patula), ‘Hurrah’ petunia (Petunia hybrida), ‘Titan’ madagascar periwinkle (Catharanthus roseus), and ‘Double Zahara’ zinnia (Zinnia marylandica). Visible injury, plant height, number of flowers, and dry weight were recorded at specific time intervals after treatment. When averaged across all annual plant species, the 1/10× rate of 2,4-D plus glyphosate resulted in 51% injury 28 days after treatment, whereas the 1/10× rate of dicamba plus glyphosate resulted in 43% injury. Treatments causing the greatest injury also resulted in the greatest reduction of dry weight, height, and flower production. Coleus was the most sensitive species in the study; dry weight was reduced by 16% and 18% compared with the nontreated controls from 1/300× rates of 2,4-D plus glyphosate and dicamba plus glyphosate, respectively. French marigold and zonal geranium had greater sensitivity to treatments containing 2,4-D, but coleus and zinnia had greater sensitivity to treatments containing dicamba. Petunia exhibited a high tolerance to 2,4-D or dicamba applied alone (>6% injury) but was highly sensitive when glyphosate was added to 2,4-D and dicamba (<65% injury). The 1/100× and 1/300× rates that are likely to equate to sublethal rates in field settings, resulted in less than 15% injury across all flower species except coleus and petunia.

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

Phytogenic Synthesis of Nano Silver from Madagascar Periwinkle Extracts and Their Angiogenic Activities in Zebrafish Embryos (ZFE)

Green biosynthesis of silver nanoparticles utilizing plant extracts is a simple, cost effective system of generating nanoparticles in an ecofriendly environment. In the present study, biosynthesis of silver nanoparticles was carried out using different parts of Madagascar periwinkle plant extracts. The production of the silver nanoparticles (AgNPs) was detected visually by change in color of the reaction mixture from yellow to reddish brown in a 8 h treatment at 37 °C. Further, the AgNPs were analyzed by various standard characterization techniques such UV-Vis spectroscopy, Electron Microscopy (TEM and SEM), FT-IR, and GC-MS analysis. The UV-Visible spectrum showed the plasmon resonance peak for AgNPs at 435 nm, indicating the formation of silver nanoparticles. FTIR analysis showed a good interaction between the plant extract and NPs. The morphology of the NPs varied in shape has observed through TEM with the particle size less than 10 nm. The phytoformulated nanoparticles were found to be well dispersed in solution and stable due to capping molecules present in the reaction mixture. The chromatographic analysis (GC-MS) displayed a prominent fragmentation pattern especially at the higher molecular weight in the range of 280 to 271. Angiogenesis is an important physiological process that plays a crucial role in several pathological conditions (tumor growth and metastasis). The green synthesized AgNPs were assessed for their angiogenic inhibition properties using zebrafish embryos as a model system. The biogenic AgNPs treatment at the dose of 100 μl and 150 μl showed significant anti-angiogenesis (inhibitory effect on angiogenesis), which represents more effectiveness in controlling the vessel formation during pathological conditions.

Effect of gamma radiation on morphological and genetic variation in regenerated plantlets Catharanthus roseus L. (G) Don

Catharanthus roseus L. (G) Don, commonly known as Madagascar periwinkle, is an important medicinal plant receiving much attention from researchers. This plant is a rich source of terpenoid indole alkaloids (TIAs). The gamma radiation and somaclonal variation may provide suitable methods for genetic changes to increase medicinal components of C. roseus. In the present study, 30 and 60 (Gy) gamma rays were irradiated on seeds. C. roseus seeds were cultured in Murashige and Skoog (MS) medium in three different groups, 0 Gy (control) 30, and 60 Gy gamma irradiations. Morphological characteristics of regenerated plantlets were measured for surface area of leaves, stem length, root length, and fresh weight. The genetic variations were evaluated using nine inter-simple sequence repeats and 25 sequences related amplified polymorphism markers. Morphological results showed a significant difference between studied groups in leaf surface and twisted leaf in 60 Gy irradiated plantlets. The analysis of molecular variance revealed the significant genetic difference among groups. STRUCTURE analysis showed genetic variability both within and among groups. Using the NJ tree analysis, the plants studied were categorized into three distinct groups, which were supported by principal coordinate analysis (PCoA). In conclusion, gamma irradiation produced morphological and genetic changes in C. roseus regenerated in vitro plantlets providing to be a suitable method for further mutation breeding in periwinkle.