Chelidonium majus Induces Apoptosis of Human Ovarian Cancer Cells via ATF3-Mediated Regulation of Foxo3a by Tip60.

Forkhead transcription factor 3a (Foxo3a) is believed to be a tumor suppressor as its inactivation leads to cell transformation and tumor development. However, further investigation is required regarding the involvement of the activating transcription factor 3 (ATF3)-mediated Tat-interactive protein 60 (Tip60)/Foxo3a pathway in cancer cell apoptosis. This study demonstrated that Chelidonium majus upregulated the expression of ATF3 and Tip60 and promoted Foxo3a nuclear translocation, ultimately increasing the level of Bcl-2-associated X protein (Bax) protein. ATF3 overexpression stimulated Tip60 expression, while ATF3 inhibition by siRNA repressed Tip60 expression. Furthermore, siRNA-mediated Tip60 inhibition significantly promoted Foxo3a phosphorylation, leading to blockade of Foxo3a translocation into the nucleus. Thus, we were able to deduce that ATF3 mediates the regulation of Foxo3a by Tip60. Moreover, siRNA-mediated Foxo3a inhibition suppressed the expression of Bax and subsequent apoptosis. Taken together, our data demonstrate that Chelidonium majus induces SKOV-3 cell death by increasing ATF3 levels and its downstream proteins Tip60 and Foxo3a. This suggests a potential therapeutic role of Chelidonium majus against ovarian cancer.

Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.

Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.

Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells.

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the upregulation of CFS (cheilanthifoline synthase) to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h respectively, under MJ elicitation. Besides, MJ upregulated the expression of TNMT (tetrahydroprotoberberine N-methyltransferase) to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs' level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

Comparative analysis of the root and leaf transcriptomes in Chelidonium majus L.

Chelidonium majus is a traditional medicinal plant, which commonly known as a rich resource for the major benzylisoquinoline alkaloids (BIAs), including morphine, sanguinarine, and berberine. To understand the biosynthesis of C. majus BIAs, we performed de novo transcriptome sequencing of its leaf and root tissues using Illumina technology. Following comprehensive evaluation of de novo transcriptome assemblies produced with five programs including Trinity, Bridger, BinPacker, IDBA-tran, and Velvet/Oases using a series of k-mer sizes (from 25 to 91), BinPacker was found to produce the best assembly using a k-mer of 25. This study reports the results of differential gene expression (DGE), functional annotation, gene ontology (GO) analysis, classification of transcription factor (TF)s, and SSR and miRNA discovery. Our DGE analysis identified 6,028 transcripts that were up-regulated in the leaf, and 4,722 transcripts that were up-regulated in the root. Further investigations showed that most of the genes involved in the BIA biosynthetic pathway are significantly expressed in the root compared to the leaf. GO analysis showed that the predominant GO domain is "cellular component", while TF analysis found bHLH to be the most highly represented TF family. Our study further identified 10 SSRs, out of a total of 39,841, that showed linkage to five unigenes encoding enzymes in the BIA pathway, and 10 conserved miRNAs that were previously not detected in this plant. The comprehensive transcriptome information presented herein provides a foundation for further explorations on study of the molecular mechanisms of BIA synthesis in C. majus.

Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.

Combination of transcriptomic and proteomic approaches helps to unravel the protein composition of Chelidonium majus L. milky sap.

MAIN CONCLUSION: A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.

Precocious progression of tissue maturation instructs basipetal initiation of leaflets in Chelidonium majus subsp. asiaticum (Papaveraceae).

PREMISE OF THE STUDY: On a compound leaf, leaflet primordia are repetitively formed along the apical-basal axis, with the direction varying among taxa. Why and how the directions vary among species is yet to be solved, although a change in a single factor was proposed to cause the variation. In this study, we compared two species in the Papaveraceae with different directions of leaflet initiation, Chelidonium majus subsp. asiaticum (basipetal) and Eschscholzia californica (acropetal). Because E. californica has been studied in some detail, we focused on C. majus and asked how basipetal pattern is achieved. • METHODS: Since only immature leaf primordial tissue has leaflet-generating competency, we performed histological and gene expression analyses on markers of the tissue maturation state. In addition, we performed a time-course analysis of leaf primordial growth. • KEY RESULTS: Quantitative reverse transcription-PCR analysis demonstrated that a putative regulator of tissue maturation in C. majus, the CINCINNATA homolog, had higher expression in apical parts than in basal parts during the organogenetic phase. In contrast, expression of the CIN homolog was not elevated in either the apical or basal parts in E. californica during the organogenetic phase. • CONCLUSIONS: In C. majus, apical parts of leaf primordia have already lost leaflet-generating competency during the organogenetic phase. We propose that precocious progression of the maturation process instructs basipetal progression of leaflet initiation in C. majus. This is not the mirror image of data on E. californica, which shows the opposite direction in leaflet formation, indicating that variation in direction is not attributable to a change in a single factor.

Expression patterns of STM-like KNOX and Histone H4 genes in shoot development of the dissected-leaved basal eudicot plants Chelidonium majus and Eschscholzia californica (Papaveraceae).

Knotted-like homeobox (KNOX) genes encode important regulators of shoot development in flowering plants. In Arabidopsis, class I KNOX genes are part of a regulatory system that contributes to indeterminacy of shoot development, delimitation of leaf primordia and internode development. In other species, class I KNOX genes have also been recruited in the control of marginal blastozone fractionation during dissected leaf development. Here we report the isolation of class I KNOX genes from two species of the basal eudicot family Papaveraceae, Chelidonium majus and Eschscholzia californica. Sequence comparisons and expression patterns indicate that these genes are orthologs of SHOOTMERISTEMLESS (STM), a class I KNOX gene from Arabidopsis. Both genes are expressed in the center of vegetative and floral shoot apical meristems (SAM), but downregulated at leaf or floral organ initiating sites. While Eschscholzia californica STM (EcSTM) is again upregulated during acropetal pinna formation, in situ hybridization could not detect Chelidonium majus STM (CmSTM) transcripts at any stage of basipetal leaf development, indicating divergent evolution of STM gene function in leaves within Papaveraceae. Immunolocalization of KNOX proteins indicate that other gene family members may control leaf dissection in both species. The contrasting direction of pinna initiation in the two species was also investigated using Histone H4 expression. Leaves at early stages of development did not reveal notable differences in cell division activity of the elongating leaf axis, suggesting that differential meristematic growth may not play a role in determining the observed dissection patterns.

[Genetic aspects of the relationship between isoquinoline alkaloids and mineral elements in greater celandine (Chelidonium majus L.)]. / Geneticheskii aspekt vzaimosviazi izokhinolinovykh alkaloidov i élementov u chistotela bol'shogo (Chelidonium majus L.).

Interrelations between the total content of isoquinoline alkaloids, concentrations of quaternary protoberberines and benzophenanthridines, and the amount of K, Cu, Co, Al, Ba, and Zn in aerial parts of individual celandine plants were revealed, within a single cenopopulation, using correlation analysis and regression analysis. Mathematical models describing the regulation of isoquinoline metabolism by some of the mineral elements were obtained in the analytical form. The results suggest that this process is genetically determined.