Inter-relationships between the heterotrimeric Gß subunit AGB1, the receptor-like kinase FERONIA, and RALF1 in salinity response.

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor-like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G-protein-coupled-receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1-like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein ß subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt-induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na+ accumulation and decreasing K+ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1-independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.

Wounding activates a 47 kDa MAP kinase in Catharanthus roseus (L.) G. Don.

Catharanthus roseus (L.) G. Don. is an important medicinal plant (Fam. Apocynaceae) known for its alkaloids that are accumulated in response to various biotic and abiotic stresses including wounding. Mitogen activated protein (MAP) kinases are important components of cellular signaling system transducing these stress stimuli into intracellular responses by phosphorylation of downstream transcription factors, regulators or inhibitors leading to expression of stress responsive genes. Here, we report the activation of a 47 kDa MAP Kinase in C. roseus in response to wounding. The immunoprecipitation coupled with in-gel kinase assay revealed tyrosine phosphorylation of the protein. Partial purification of the MAP kinase was also attempted. We observed a size variation for the kinase when purified using different schemes.

Clade IVa Basic Helix-Loop-Helix Transcription Factors Form Part of a Conserved Jasmonate Signaling Circuit for the Regulation of Bioactive Plant Terpenoid Biosynthesis.

Plants produce many bioactive, specialized metabolites to defend themselves when facing various stress situations. Their biosynthesis is directed by a tightly controlled regulatory circuit that is elicited by phytohormones such as jasmonate (JA). The basic helix-loop-helix (bHLH) transcription factors (TFs) bHLH iridoid synthesis 1 (BIS1) and Triterpene Saponin Activating Regulator (TSAR) 1 and 2, from Catharanthus roseus and Medicago truncatula, respectively, all belong to clade IVa of the bHLH protein family and activate distinct terpenoid pathways, thereby mediating monoterpenoid indole alkaloid (MIA) and triterpene saponin (TS) accumulation, respectively, in these two species. In this study, we report that promoters of the genes encoding the enzymes involved in the specific terpenoid pathway of one of these species can be transactivated by the orthologous bHLH factor from the other species through recognition of the same cis-regulatory elements. Accordingly, ectopic expression of CrBIS1 in M. truncatula hairy roots up-regulated the expression of all genes required for soyasaponin production, resulting in strongly increased levels of soyasaponins in the transformed roots. Likewise, transient expression of MtTSAR1 and MtTSAR2 in C. roseus petals led to up-regulation of the genes involved in the iridoid branch of the MIA pathway. Together, our data illustrate the functional similarity of these JA-inducible TFs and indicate that recruitment of defined cis-regulatory elements constitutes an important aspect of the evolution of conserved regulatory modules for the activation of species-specific terpenoid biosynthesis pathways by common signals such as the JA phytohormones.

Investigation of the chemomarkers correlated with flower colour in different organs of Catharanthus roseus using NMR-based metabolomics.

INTRODUCTION: Flower colour is a complex phenomenon that involves a wide range of secondary metabolites of flowers, for example phenolics and carotenoids as well as co-pigments. Biosynthesis of these metabolites, though, occurs through complicated pathways in many other plant organs. The analysis of the metabolic profile of leaves, stems and roots, for example, therefore may allow the identification of chemomarkers related to the final expression of flower colour. OBJECTIVE: To investigate the metabolic profile of leaves, stems, roots and flowers of Catharanthus roseus and the possible correlation with four flower colours (orange, pink, purple and red). METHODS: (1) H-NMR and multivariate data analysis were used to characterise the metabolites in the organs. RESULTS: The results showed that flower colour is characterised by a special pattern of metabolites such as anthocyanins, flavonoids, organic acids and sugars. The leaves, stems and roots also exhibit differences in their metabolic profiles according to the flower colour. Plants with orange flowers featured a relatively high level of kaempferol analogues in all organs except roots. Red-flowered plants showed a high level of malic acid, fumaric acid and asparagine in both flowers and leaves, and purple and pink flowering plants exhibited high levels of sucrose, glucose and 2,3-dihydroxy benzoic acid. High concentrations of quercetin analogues were detected in flowers and leaves of purple-flowered plants. CONCLUSIONS: There is a correlation between the metabolites specifically associated to the expression of different flower colours and the metabolite profile of other plant organs and it is therefore possible to predict the flower colours by detecting specific metabolites in leaves, stems or roots. This may have interesting application in the plant breeding industry.

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

Slowly developing drought stress increases photosynthetic acclimation of Catharanthus roseus.

Our understanding of plant responses to drought has improved over the decades. However, the importance of the rate of drought imposition on the response is still poorly understood. To test the importance of the rate at which drought stress develops, whole-plant photosynthesis (P(net) ), respiration (R(dark) ), daily carbon gain (DCG), daily evapotranspiration (DET) and water use efficiency (WUE) of vinca (Catharanthus roseus), subjected to different drought imposition rates, were investigated. We controlled the rate at which the substrate dried out with an automated irrigation system that allowed pot weight to decrease gradually throughout the drying period. Fast, intermediate and slow drying treatments reached their final pot weight [500 g, substrate water content (θ) ≈ 0.10 m³ m(-3) ] after 3.1, 6.6 and 10 days, respectively. Although all drying treatments decreased P(net) and R(dark) , slow drying reduced P(net) and R(dark) less than fast drying. At a θ < 0.10 m³ m(-3) , DCG and DET in the slow drying treatment were reduced by ≈50%, whereas DCG and DET in the fast drying treatment were reduced by 85 and 70% at a θ of 0.16 m(3) m(-3) . Plants exposed to slow drought imposition maintained a high WUE, even at θ < 0.10 m³ m(-3) . Overall, physiological responses to low θ were less severe in plants subjected to slow drying as compared with fast drying, even though the final θ was lower for plants exposed to slow drying. This suggests that the rate at which drought stress develops has important implications for the level of acclimation that occurs.

Production and metabolic engineering of terpenoid indole alkaloids in cell cultures of the medicinal plant Catharanthus roseus (L.) G. Don (Madagascar periwinkle).

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a plant species known for its production of TIAs (terpenoid indole alkaloids), many of which are pharmaceutically important. Ajmalicine and serpentine are prescribed for the treatment of hypertension, whereas the bisindoles vinblastine, vincristine and 3',4'-anhydrovinblastine are used for their antineoplastic activity in the treatment of many cancers. However, TIAs are produced in small yields in C. roseus, which make them expensive. Cell and metabolic engineering has focused on increasing flux through the TIA pathway by various means, including optimization of medium composition, elicitation, construction of noval culture systems and introduction of genes encoding specific metabolic enzymes into the C. roseus genome. The present review will attempt to present the state-of-the-art of research in this area and provide an update on the cell and metabolic engineering of TIAs in C. roseus. We hope that this will contribute to a better understanding of the ways in which TIA production can be achieved in different C. roseus culture systems.

Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time.

Jasmonic acid (JA) activates the transcriptional regulator ORCA3, which has a role in regulating the terpenoid indole alkaloid (TIA) pathway within Catharanthus roseus. The TIA pathway leads to the production of the anticancer drugs vinblastine and vincristine. This work explores the transient effects of overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter system in C. roseus hairy roots along with the simultaneous feeding of JA. The changes in TIA metabolites and in mRNA transcripts of pathway genes and regulators were tracked for 72h. Upon induction of ORCA3 expression and elicitation with JA, ORCA3 transcripts increased 170-fold whereas ORCA3 expression caused an 89-fold increase and JA elicitation caused a 5-fold increase in ORCA3 transcripts. JA treatment caused the largest increase in TIA metabolites and transcripts of pathway genes. These transcripts displayed a transient response with the maximum expression reached between 12 and 24h. In the samples overexpressing ORCA3, the largest increase in the transcripts of ZCT1 and ZCT2 (ZCT-zinc finger-binding protein), TIA transcriptional repressors, coincided with the largest increase in ORCA3 transcripts. This counter response of transcriptional repressors may explain why the large increase in ORCA3 transcripts do not correspond with larger increases in transcripts of TIA pathway genes.

Endogenous hormonal and enzymatic responses of Catharanthus roseus with triadimefon application under water deficits.

The effect of triadimefon was investigated in a medicinal plant, Catharanthus roseus subjected to water deficit stress. The abscisic acid (ABA) level, DNA and RNA contents and activities of ATPase and protease were found varying in different parts of the plants under treatment. Drought treatment increased the ABA level more than twofold in all parts of the plants. TDM treatment to the drought stressed plants showed highest contents. In roots, stem and leaves, drought stress caused a decrease in the DNA and RNA contents when compared with control and other treatments. TDM treatment with drought increased the nucleic acid contents to the level of the control roots. The activity of ATPase and protease were increased under drought treatment and lowered due to TDM applications. This information could be useful in the field of soil water deficits reclamation efforts by using plant growth regulators.

Growth and photosynthetic pigments responses of two varieties of Catharanthus roseus to triadimefon treatment.

Triadimefon, potential fungicide cum plant-growth retardant was used in this study to investigate its effect on the growth and the photosynthetic pigment contents of two varieties of Catharanthus roseus (L.) G. Don. The plants of both varieties were subjected to 15 mg l(-1) triadimefon treatment by soil drenching 30, 45, 60, and 75 days after planting (DAP). Plants were uprooted on 90 DAP, and morphological parameters, like plant height, number of leaves, leaf area, root length and fresh and dry weights were determined. The photosynthetic pigments, like chlorophylls a and b, total chlorophyll, carotenoids, floral pigment, anthocyanin, were extracted and estimated. It was observed that plant height, number of leaves and leaf area were decreased and that root length, fresh and dry weights were increased under triadimefon treatment. The photosynthetic and floral pigments were increased under triadimefon treatment in both varieties. The results suggest that the application of this plant-growth retardant (triadimefon) has favourable effects on the reduction of plant height; it can thus be used for replacing manual hand pruning and for improving floral and vegetation colour in bedding plants like C. roseus.

Differential responses in water use efficiency in two varieties of Catharanthus roseus under drought stress.

Two varieties, rosea and alba, of Catharanthus roseus (L.) G. Don. were screened for their water use efficiency under two watering regimes, viz. 60 and 100% filed capacity in the present study. Drought stress was imposed at 60% filed capacity from 30 to 70 days after sowing, while the control pots were maintained at 100% filed capacity throughout the entire growth period. Leaf area duration, cumulative water transpired, water use efficiency, net assimilation rate, mean transpiration rate, harvest index, biomass and yield under the water deficit level were measured from both stressed and well-watered control plants. Water use efficiency significantly increased in both varieties under water stress. Drought stress decreased leaf area duration, cumulative water transpired, net assimilation rate, mean transpiration rate, harvest index, and biomass yield in both varieties studied. Among the varieties, rosea variety showed the best results.

Alterations in lipid peroxidation, electrolyte leakage, and proline metabolism in Catharanthus roseus under treatment with triadimefon, a systemic fungicide.

Triadimefon (TDM), a systemic fungicide with non-traditional plant-growth regulator properties, was administered to Catharanthus roseus (L.) G. Don. plants in order to determine its effects on oxidative injury in terms of H2O2 content, lipid peroxidation (LPO), electrolyte leakage (EL), protein and amino acid contents, as well as proline metabolism. The LPO, estimated as thiobarbituric acid-reactive substances (TBARS), decreased under TDM treatment. It was found that H2O2 and EL were reduced under TDM treatment when compared to control. TDM treatment caused a significant increase in the protein and amino acid contents. Glycine betaine (GB) and proline (PRO) significantly accumulated in C. roseus under stress arisen from fungicide applications. Proline oxidase (PROX) activities reduce the PRO content and gamma-glutamyl kinase (gamma-GK) accelerates the synthesis of PRO. Under TDM treatment, the activity of PROX decreased and the gamma-GK activity increased. From our results, it is suggested that fungicide triadimefon causes activation of metabolic processes in the medicinal plant Catharanthus roseus. These findings are of great significance for the cultivation of this medicinal plant, as it was previously reported that TDM causes an enhancement of antioxidant metabolism and ajmalicine production in C. roseus.

Potential of Vinca rosea extracts in modulating trace element profile: a chemopreventive approach.

Diethylnitrosamine (DEN) was used as cancer-inducing agent in the experimental animals. Vinca rosea extract was supplemented with the drinking water as a chemopreventive agent. After 4 wk of treatment, animals were sacrificed and livers were excised. Nuclei and mitochondria were separated by differential centrifugation. The proton-induced X-ray emission technique has been used as the analytical method. Elemental analysis were performed for whole liver, nuclei, and mitochondria.V. rosea plant parts were also analyzed for elemental contents. Treatment with DEN caused an increase of Ni, Zn, and Cr levels in the whole liver and nuclei. There is an increase in Fe concentration in the liver, although the level decreased in mitochondria. The concentrations of Br and Ca were unchanged in the liver as a whole, but there were substantial increases of Br in nuclei and mitochondria, whereas Ca levels depleted drastically in these two organelles. Vinca extracts were effective in reverting the changes in the elemental concentration in the hepatic tissue as a whole, but were not that effective at subcellular levels.

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots.

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.

Calcium chloride effects on salinity-induced oxidative stress, proline metabolism and indole alkaloid accumulation in Catharanthus roseus.

Catharanthus roseus (L.) G. Don. plants were grown with NaCl and CaCl2 in order to study the effect of CaCl2 on NaCl-induced oxidative stress in terms of lipid peroxidation (TBARS content), H2O2 content, osmolyte concentration, proline (PRO)-metabolizing enzymes, antioxidant enzyme activities, and indole alkaloid accumulation. The plants were treated with solutions of 80 mM NaCl, 80 mM NaCl with 5 mM CaCl2 and 5 mM CaCl2 alone. Groundwater was used for irrigation of control plants. Plants were uprooted randomly on 90 days after sowing (DAS). NaCl-stressed plants showed increased TBARS, H2O2, glycine betaine (GB) and PRO contents, decreased proline oxidase (PROX) activity, and increased gamma-glutamyl kinase (gamma-GK) activity when compared to control. Addition of CaCl2 to NaCl-stressed plants lowered the PRO concentration by increasing the level of PROX and decreasing the gamma-GK activities. Calcium ions increased the GB contents. CaCl2 appears to confer greater osmoprotection by the additive role with NaCl in GB accumulation. The antioxidant enzymes superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased under salinity and further enhanced due to CaCl2 treatment. The NaCl-with-CaCl2-treated C. roseus plants showed an increase in total indole alkaloid content in shoots and roots when compared to NaCl-treated and untreated plants.

Responses of transformed Catharanthus roseus roots to femtomolar concentrations of salicylic acid.

Catharanthus roseus transformed roots were cultured in the presence of salicylic acid (SA) at concentrations between 0.1 fM and 100 pM and the effect on root growth was evaluated. Significant morphological changes in the lateral roots were recorded on day two in the SA treatment. Presence of SA increased root cap size and caused the appearance of lateral roots closer to the root tip. The bioassay was sensitive enough to allow testing of low concentrations of other growth regulators that may affect root morphology and physiology.

Rapid and non-invasive detection of plants senescence using a delayed fluorescence technique.

Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II in a plant is considered to be the primary site where delayed fluorescence (DF) is produced. We report here a simple, rapid, and non-invasive technique for detecting plants senescence based on quantitative measurements of DF. In the experimental study, various senescence symptoms induced by age or hormones were examined in the Catharanthus roseus L. G. Don plants. Detecting the DF emissions from leaves with a home-made DF biosensor enables DF parameters of C. roseus to be produced in a short time. Meanwhile, evaluations of leaves senescence were made from measurements of chlorophyll content, ion leakage, and net photosynthesis rate (Pn) based on the consumption of CO2 in the tested plants. The results of our investigation demonstrate that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content during age-dependent and hormone-modulated senescence. Moreover, the DF intensity negatively correlates with ion leakage in both types of senescence. With proper calibration, DF may provide an important approach for monitoring senescence process in vivo and quantitatively evaluating senescence extent. Therefore, a DF technique could be potentially useful for less time-consuming and automated screening of the interesting mutants with genetic modifications that change the plant senescence progress.

Transcription factor Agamous-like 12 from Arabidopsis promotes tissue-like organization and alkaloid biosynthesis in Catharanthus roseus suspension cells.

In Catharanthus roseus, monomeric terpenoid indole alkaloids (TIAs) are biosynthesized in specific tissues, particularly in roots, but failed to be produced by in vitro undifferentiated suspension cells. In this paper, we describe the impact of the root-specific MADS-box transcription factor Agamous-like 12 (Agl12) from Arabidopsis thaliana on the differentiation of suspension cells from C. roseus. The expression of Agl12 is sufficient to promote an organization of suspension cells into globular parenchyma-like aggregates but is insufficient by itself to induce complete morphological root differentiation. Agl12 expression selectively increases the expression of genes encoding enzymes involved in the early biosynthesis steps of the terpenic precursor of alkaloids. The transgenic cell lines expressing Agl12 produced significant amounts of ajmalicine, an antihypertensive TIA that normally accumulates in C. roseus roots. The present paper indicates that transcription factors involved in tissue or organ differentiation may constitute new metabolic engineering tools that could help to design in vitro cultured cells able to produce specific valuable secondary metabolites.

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root.

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.

Magnetic field exposure stiffens regenerating plant protoplast cell walls.

Single suspension-cultured plant cells (Catharanthus roseus) and their protoplasts were anchored to a glass plate and exposed to a magnetic field of 302 +/- 8 mT for several hours. Compression forces required to produce constant cell deformation were measured parallel to the magnetic field by means of a cantilever-type force sensor. Exposure of intact cells to the magnetic field did not result in any changes within experimental error, while exposure of regenerating protoplasts significantly increased the measured forces and stiffened regenerating protoplasts. The diameters of intact cells or regenerating protoplasts were not changed after exposure to the magnetic field. Measured forces for regenerating protoplasts with and without exposure to the magnetic field increased linearly with incubation time, with these forces being divided into components based on the elasticity of synthesized cell walls and cytoplasm. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye, and no changes were noted after exposure to the magnetic field. Analysis suggested that exposure to the magnetic field roughly tripled the Young's modulus of the newly synthesized cell wall without any lag.