Roseus Seedlings Research Articles

A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon-VIGS) approach to study specialized metabolism in medicinal plants

BackgroundVirus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites.ResultsWe evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents.ConclusionsThe widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.

Isolation of Bacterial Endophytes Associated with Cinchona ledgeriana Moens. and Their Potential in Plant-growth Promotion, Antifungal and Quinoline Alkaloids Production.

For centuries, quinoline alkaloids from the tree bark of Cinchona ledgeriana (C. ledgeriana) have been used in the treatment of malaria. However, unsustainable harvesting and poor growth conditions greatly limit its use as raw materials. Since plant endophytes are known to contribute to the physiology of the host and its metabolism for survival, this study showed the potential of endophytes isolated from C. ledgeriana roots in promoting the germination of Catharathus roseus (C. roseus) seedlings and the biosynthesis of quinoline alkaloid. In this present study, we found that the Enterobacteriaceae family comprised the majority of the bacterial community, with Klebsiella pneumoniae being the most abundant species at the C. ledgeriana roots. Characterization of culturable bacterial endophytes from the C. ledgeriana roots showed that all the isolates displayed plant growth-promoting factors and antifungal activities. Interestingly, chromatographic analyses led to the identification of the quinoline alkaloids producing Achromobacter xylosoxidans (A. xylosoxidans) A1. Moreover, the co-cultures of A. xylosoxidans A1, Cytobacillus solani (C. solani) A3, and Klebsiella aerogenes A6 increased the fresh and dry weight of the C. roseus seedlings. These results suggest that these bacterial endophytes may enhance quinine and quinidine production as well as the growth of the plant host.

Hishimonus phycitis: Possible Vector of 16SrI-B Subgroup Phytoplama

Witches-broom symptoms of Catharanthus roseus are caused by ‘Candidatus phytoplasma is a terrible disease in the Uttar Pradesh State of India. The disease has been transmitted to infected C. roseus seedlings by grafting and cutting of stems. Heavy infestation of Hishimonus phycitis was recorded in ornamental garden during rainy season with witches- broom and phyllody disease of C. roseus at surveyed location of Shahjahanpur, Uttar Pradesh. To confirm the phytoplasma in C. roseus and the identification of phytoplasma causing insect vector, DNA were isolated from both of the infected plants and suspected H. phycitis. Isolated DNA of both insects and plants were performed to PCR assay with the help of phytoplasma specific universal primer P1/P7 and nested PCR with R16F2n/R2 which yielded about 1.2 kb amplicon in both the H. phycitis and witches-broom symptomatic samples. Both suspected amplified DNA samples from C. roseus and H. phycitis were sequenced. BLASTn and RFLP analysis were performed for the sequence alignment and subgroup assignment. BLASTn analyses were produces a result of 100% sequence similarity among themselves and 99% similarity with strain of 16SrI group whereas RFLP produces 16SrI-B subgroup. The study indicated that H. phycitis may play role as a vector in the transmission of these phytoplasma in C. roseus crops.

GATA and Phytochrome Interacting Factor Transcription Factors Regulate Light-Induced Vindoline Biosynthesis in Catharanthus roseus.

Catharanthus roseus is the exclusive source of an array of terpenoid indole alkaloids including the anticancer drugs vincristine and vinblastine, derived from the coupling of catharanthine and vindoline. Leaf-synthesized vindoline is regulated by light. A seven-step enzymatic process is involved in the sequential conversion of tabersonine to vindoline; however, the regulatory mechanism controlling the expression of genes encoding these enzymes has not been elucidated. Here, we identified CrGATA1, an Leu-Leu-Met domain GATA transcription factor that regulates light-induced vindoline biosynthesis in C. roseus seedlings. Expression of CrGATA1 and the vindoline pathway genes T16H2, T3O, T3R, D4H, and DAT was significantly induced by light. In addition, CrGATA1 activated the promoters of five light-responsive vindoline pathway genes in plant cells. Two GATC motifs in the D4H promoter were critical for CrGATA1-mediated transactivation. Transient overexpression of CrGATA1 in C. roseus seedlings resulted in up-regulation of vindoline pathway genes and increased vindoline accumulation. Conversely, virus-induced gene silencing of CrGATA1 in young C. roseus leaves significantly repressed key vindoline pathway genes and reduced vindoline accumulation. Furthermore, we showed that a C. roseus Phytochrome Interacting Factor, CrPIF1, is a repressor of CrGATA1 and vindoline biosynthesis. Transient overexpression or virus-induced gene silencing of CrPIF1 in C. roseus seedlings altered CrGATA1 and vindoline pathway gene expression in the dark. CrPIF1 repressed CrGATA1 and DAT promoter activity by binding to G/E-box/PBE elements. Our findings reveal a regulatory module involving Phytochrome Interacting Factor -GATA that governs light-mediated biosynthesis of specialized metabolites.

The miRNAome of Catharanthus roseus: identification, expression analysis, and potential roles of microRNAs in regulation of terpenoid indole alkaloid biosynthesis

MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites.

Low Light Affacting Alkaloids Accumulation and Related Biosynthetic Pathway Genes Expression in Leaves of <i>Catharanthus roseus</i> Seedlings

The alkaloids contents were investigated by High performance liquid chromatography (HPLC) in Catharanthus roseus aseptic seedlings leaves, the expressions of terpenoid indole alkaloids (TIAs) biosynthesis pathway genes were detected by semi-quantitative RT-PCR. The plastic films treatment induces a low light condition. HPLC analysis shows, the contents of vindoline (VIN) and catharanthine (CAT) are slight increase first and significant decrease (p<0.05) under plastic films, the vinblastine (VBL) content increase and reach the maximum (0.028 mg g-1±0.0051) on the 15th day of treatment. TIA biosynthesis pathway genes expression change under low light. The correlation analysis indicates, VIN accumulation is significantly correlated with the gene expression of desacetoxyvindoline-4-hydroxylase (D4h) and deacetylvindoline-4-O-acetyl transferase (Dat), and CAT accumulation is significantly correlated with strictosidine synthase (Str) gene expression (p<0.05). The low light treatment with plastic films accelerates the accumulation of VBL in C. roseus seedlings leaves.

外源乙烯利、锌协同促进长春花叶片中长春碱的含量

药用植物长春花叶片中含有的长春碱在外源锌胁迫条件下其含量有一定程度上的增加。而在此基础上加入外源乙烯利后的协同作用下，更加促进了长春碱含量的增加，并且随着处理浓度的增加使之呈现出规律性变化。在外源锌和乙烯利的协同作用条件下，长春花叶片的干物质积累以及乙烯气体释放量等参数也有显著提高。 Under Zinc (Zn) stress conditions, the production of vinblastine was increased in the medicinal plant Catharanthus roseus leaves. The combinational treatment with Zn and ethephon further promoted vinblastine accumulation in C. roseus leaves compared with Zn-stressed plants. This elevation of vinblastine induced by ethephon was dependent on the dose of exogenous ethephon. Besides, the dry biomass accumulation of leaves and ethylene release were also increased significantly in Catharanthus roseus seedlings affected by the combined treatment with exogenous Zn and ethephon.

Endophytic Methylobacterium extorquens expresses a heterologous β-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Methyl jasmonic acid induced expression pattern of terpenoid indole alkaloid pathway genes in Catharanthus roseus seedlings

Madagascar periwinkle (Catharanthus roseus) produces diverse terpenoid indole alkaloids (TIAs), including the anti-cancer drugs vincristine and vinblastine. In this study, the effects of methyl jasmonic acid (MeJA) treatment on TIA metabolism and gene expression in seedlings were analyzed. Several reference genes were also isolated from periwinkle. These reference genes, as well as ribosomal protein (RSP9) and cyclophilin (CYC) genes, were characterized to determine which are most suitable for use as expression profiling control genes. The results show that TIA genes exhibit significant variation in the magnitude and timing of induction by MeJA. ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor gene, exhibited the greatest increase in transcript levels, with increases up to 25 fold observed 0.5 h after MeJA treatment. MeJA-induced increases in transcript levels occurred in the following order: ORCA3, desacetoxyvindoline 4-hydroxylase (D4H), strictosidine synthase (STR), tryptophan decarboxylase (TDC), geraniol 10-hydroxylase (G10H) and cytochrome P-450 reductase (CPR). The results suggest that variations in the timing of MeJA induced increases in TIA transcript levels might be related to complex interactions between different transcription factors, such as ORCA3, and other factors. CrEF1α and CrUBQ11 are the most stable genes out of the 8 tested under the conditions of these experiments.

Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for paratransgenic control strategy of Citrus variegated chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.

Alkaloid variations in Catharanthus roseus seedlings treated by different temperatures in short term and long term

A study was conducted to investigate the effects of high temperature on variations of alkaloid metabolism in C. roseus seedlings in Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Heilongiang, China. 60-day-old C. roseus seedlings with 3–4 pairs of leaves were incubated in chambers with temperature of 30°C and 40°C for short-term heat shock experiment and 20°C, 25°C and 35°C for long-term experiment. The contents of vindoline, catharanthine, vinblastine and vincristine in C. roseus leaves and root were checked at different temperatures in short term (1–6 h) and long term (1–16 d). Results showed that under short-term heat shock, the contents of vindoline, catharanthine and vinblastine in leaves of the seedlings were higher at 40°C than at 30°C, but after 6 h treatment, the contents of vindoline and catharanthine under the two temperatures came to the same level. Catharanthine was exclusively distributed in C. roseus roots and its content was increased by 40% after two hours incubation at 40°C, while increased slowly at 30°C incubation and reached the highest value at 6 h. In the Long-term experiment, concentrations of monomeric alkaloids catharanthine and vindoline were higher at 20°C than at 25°C and had a sharp increase under the condition of 35°C. While for dimeric alkaloid, it showed that the higher the temperature, the earlier the peak value of vinblastine content appears. Vincristine had a continuous enhancement and attained 0.027 mg·g−1 at 16th day under 35°C condition which was higher than those in the other conditions. It was concluded that high temperature could promote the accumulation of different alkaloids in C. roseus and the accumulation characteristic is highly related to treatment time.

Accumulation of monoterpenoid indole alkaloids in periwinkle seedlings (Catharanthus roseus) as a model for the study of plant–environment interactions

Alkaloids are part of the chemical arsenal designed to protect plants against an adverse environment. Therefore, their synthesis and accumulation are frequently induced in response to certain environmental conditions and are mediated by chemical signals, which are formed as the first responses to the external stimulus. A set of experiments using Catharanthus roseus seedlings is presented to illustrate this interaction. Using the well characterized induction of monoterpenoid indole alkaloid synthesis in response to methyl jasmonate, one chemical mediator; simple and reproducible experiments are proposed to demonstrate this type of plant-environment interaction to plant physiology or biochemistry B.Sc. students. After exposure to the inducer, seedlings are analyzed for alkaloid accumulation and for the increase of tryptophan decarboxylase (TDC) enzyme activity by means of thin layer chromatography, followed by UV spectrophotometry. Alkaloids should increase as a result of the exposure to the inducer, as well as TDC activity, which plays a critical role in channeling carbon skeletons from primary to secondary metabolism in this plant.

Effects of tip-pruning treatment on source-sink regulation of Catharanthus roseus seedlings

Fifty cultivated Catharanthus roseus seedlings were selected for tip-pruning treatment and the effects of tip-pruning on seedling growth and source-sink regulation were investigated for revealing physiological mechanisms of plants. The results showed that tip-pruning treatment resulted in obvious inhibition of apical dominance and enhancement of branching numbers. The contents of soluble sugars, acid sucrose invertase activity (AI) had a great change in differently positional leaves of the seedling. The sink strength in tip leaves of seedlings dramatically declined after tip-pruning treatment, while that in the leaves at the middle and bottom of seedlings had no obvious changes. The inhibition of apical dominance of tip leaves of seedlings was caused by the diminished sink strength due to tip-pruning treatment.

Alkaloid metabolism in wounded Catharanthus roseus seedlings

The effect of mechanical wounding on alkaloid metabolism was analyzed in Catharanthus roseus seedlings. Wounding induced an increase in ajmalicine accumulation, whereas catharanthine remained unaffected. A positive dual effect on vindoline was noticed. Short and mid-term effects were detected between 12 and 24 h after mechanical damage was inflicted, and apparently resulted from the accelerated transformation of the tabersonine intermediaries to vindoline. Long-term effects involved a generalized increase in carbon flux towards alkaloid synthesis. Exposure to ethylene (1 ppm) produced similar results to those observed in wounded seedlings, suggesting that it might be mediating the wound-induced increase in alkaloid synthesis. No synergistic or additive effects were observed when wounded seedlings were exposed to ethylene or jasmonate.

The Association of Phytoplasma with Stunting, Leaf Necrosis and Witches’ Broom Symptoms in Magnolia Plants

In 1999–2000 a severe disease was observed on plants of four Magnolia spp. cultivated in a commercial nursery in Poland. Affected plants showed a progressive loss of vigour, were stunted, and had severely malformed leaves, leaf necrosis and witches’ broom. Phytoplasma was detected in magnolias with severe symptoms and in dodder‐inoculated Catharanthus roseus seedlings by nested polymerase chain reaction (PCR) assay with primer pair R16F1/R0 followed by universal (rA/fA) and group specific (R16(I)F1/R1) primer pairs which amplified a fragment of phytoplasma 16S rDNA. The PCR products (560 bp or 1.1 kb) of all samples used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes AluI and MseI produced the same profile which corresponded to that of an aster yellows phytoplasma reference strain. Phytoplasma DNA was detected throughout the growing season in roots, stems and young but not mature leaves. Electron microscope examination of the ultra‐thin sections of the leaf and stem of diseased magnolias showed collapsed and degenerated sieve tube elements with wall thickening. The reduced lumen of these sieve elements contained numerous vesicles and membrane‐bound structures, but no typical phytoplasma cells. This is the first report of aster yellows phytoplasma in magnolia identified by molecular assays.

Alkaloid repartition and DMSO effects on alkaloid cccumulation in Catharanthus seedlings

Summary Many studies have addressed the improvement of indole alkaloid production in Catharanthus roseus cell suspension culture, but only few of them were concerned with entire plants cultured in vitro . The aim of this work was to investigate alkaloid distribution and the influence of DMSO (dimethyl sulfoxide) on alkaloid accumulation in C. roseus seedlings grown in vitro . Thirty-day-old seedlings were cultured on hormone-free medium supplemented with DMSO and analysed by HPLC and TLC. An «abnormal» alkaloid distribution in seedling roots was characterised by the lack of catharanthine and the presence of yohimbine for the first time. Both ajmalicine and serpentine were also observed. Vindoline and catharanthine were detected in the aerial parts. The results with DMSO showed a decrease in alkaloid accumulation in both the roots and aerial parts.

Signalling molecules and the synthesis of alkaloids in Catharanthus roseus seedlings

The Madagascar periwinkle, Catharanthus roseus, produces numerous alkaloids, several of which have important pharmaceutical uses. Catharanthus seedlings rapidly accumulate the monomeric alkaloids, vindoline, catharanthine and tabersonine, during germination. Various plant signalling molecules were tested for their ability to enhance alkaloid synthesis in Catharanthus seedlings. The compounds tested included plant hormones, fatty acid-derived messengers and agents that can induce systemic-acquired resistance in plants. Of these compounds, only methyl jasmonate (MeJa) enhanced the synthesis of monomeric alkaloids. However, feeding of a MeJa biosynthetic precursor, or inhibition of the lipoxygenase pathway, had no effect on alkaloid production in the seedlings. We conclude that, although MeJa is able to enhance alkaloid synthesis when supplied exogenously, the lipoxygenase pathway probably does not play a role in the regulation of alkaloid synthesis during normal germination of Catharanthus. Furthermore, it was found that Catharanthus seedlings accumulate small quantities of the dimeric alkaloid, vinblastine, a valuable antitumour drug. Contrary to its effect on the accumulation of monomeric alkaloids, MeJa did not influence the accumulation of vinblastine.

Purification and partial characterization of microsomal cytochrome b 555 from the higher plant [formula omitted

Microsomal b-type hemoprotein designated, cytochrome b 555 of C. roseus seedlings was solubilized using detergents and purified by a combination of ion exchange chromatography and gel filtration to a specific content of 18.5 nmol per mg of protein. The purified cytochrome b 555 was homogeneous and estimated to have an apparent molecular weight of 16500 on SDS-PAGE. The absorption spectrum of the reduced form has major peaks at 424, 525 and 555 nm. The α-band of the reduced form is asymmetric with a pronounced shoulder at 559 nm. The spectrum of the pyridine ferrohemochrome shows absorption peaks at 557, 524 and 418 nm indicating that the cytochrome has protoheme prosthetic group. The purified cytochrome is autoxidizable and does not combine with carbon monoxide, azide or cyanide. It is reducible by NADH in the presence of NADH-cytochrome b 555 reductase partially purified from C. roseus microsomes.