Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor ß (TGFß) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences.

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

Identification and Characterization of HIRIP3 as a Histone H2A Chaperone.

HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.

Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes.

While hundreds of starch- and glycogen-degrading enzymes have been characterized experimentally in historical families such as GH13, GH14, GH15, GH57 and GH126 of the CAZy database (www.cazy.org), the α-amylase from Bacillus circulans is the only enzyme that has been characterized in family GH119. Since glycosidase families have been shown to often group enzymes with different substrates or products, a single characterized enzyme in a family is insufficient to extrapolate enzyme function based solely on sequence similarity. Here we report the rational exploration of family GH119 through the biochemical characterization of five GH119 members. All enzymes shared single α-amylase specificity but display distinct product profile. We also report the first kinetic constants in family GH119 and the first experimental validation of previously predicted catalytic residues in family GH119, confirming that families GH119 and GH57 can be grouped in the novel clan GH-T of the CAZy database.

Investigation and drug design for novel molecules from natural products as inhibitors for controlling multiple myeloma disease using in-silico tools.

Multiple myeloma (MM) is a disease that causes plasma cell growth in the bone marrow and immune globulin buildup in blood and urine. Despite recent advances in MM therapy, many still die due to its high mortality rate. A study using computational simulations analyzed 100 natural ingredients from the SANC database to determine if they inhibited the IgH domain, a known cause of multiple myeloma. Natural component Diospyrin inhibited the IgH enzyme with the best binding energy of -10.3 kcal/mol and three carbon-hydrogen bonds, followed by Parviflorone F complex with a binding energy of -10.1 kcal/mol and two conventional-hydrogen bonds. As a result, the Molecular Dynamic simulation was used to test the stability of the two complexes. During the simulation, the Diospyrin molecule dissociated from the protein at roughly 67.5 ns, whereas the Parviflorone F molecule stayed attached to the protein throughout. The latter was the subject of the investigation. The analysis of the production run data revealed that the Parviflorone F molecule exhibits a variety of conformations within the binding pocket while keeping a relatively constant distance from the protein's center of mass. The analysis of the production run data revealed that the Parviflorone F molecule exhibited a variety of conformations within the binding pocket while keeping a relatively constant distance from the protein's center of mass. The root mean square deviation (RMSD) plots for both the protein and complex showed a stable and steady average value of 4.4 Å for the first 82 nanoseconds of manufacture. As a result, the average value increased to 8.3 Å. Furthermore, the components of the binding free energy, as computed by MM-GBSA, revealed that the mean binding energy of the Parviflorone F molecule was -23.88 kcal/mol. Finally, after analyzing all of the examination data, Parviflorone F was identified as a powerful inhibitor of the IgH domain and hence of the MM disease, which requires further in-vivo conformation.Communicated by Ramaswamy H. Sarma.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.

BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Early Fruit Development Regulation-Related Genes Concordantly Expressed with TCP Transcription Factors in Tomato (Solanum lycopersicum).

The tomato (Solanum lycopersicum L.) is considered one of the most important vegetable crops globally, both agronomically and economically; however, its fruit development regulation network is still unclear. The transcription factors serve as master regulators, activating many genes and/or metabolic pathways throughout the entire plant life cycle. In this study, we identified the transcription factors that are coordinated with TCP gene family regulation in early fruit development by making use of the high-throughput sequencing of RNA (RNAseq) technique. A total of 23 TCP-encoding genes were found to be regulated at various stages during the growth of the fruit. The expression patterns of five TCPs were consistent with those of other transcription factors and genes. There are two unique subgroups of this larger family: class I and class II TCPs. Others were directly associated with the growth and/or ripening of fruit, while others were involved in the production of the hormone auxin. Moreover, it was discovered that TCP18 had an expression pattern that was similar to that of the ethylene-responsive transcription factor 4 (ERF4). Tomato fruit set and overall development are under the direction of a gene called auxin response factor 5 (ARF5). TCP15 revealed an expression that was in sync with this gene. This study provides insight into the potential processes that help in acquiring superior fruit qualities by accelerating fruit growth and ripening.

CRISPR-based systems for sensitive and rapid on-site COVID-19 diagnostics.

The COVID-19 pandemic has strained healthcare systems. Sensitive, specific, and timely COVID-19 diagnosis is crucial for effective medical intervention and transmission control. RT-PCR is the most sensitive/specific, but requires costly equipment and trained personnel in centralized laboratories, which are inaccessible to resource-limited areas. Antigen rapid tests enable point-of-care (POC) detection but are significantly less sensitive/specific. CRISPR-Cas systems are compatible with isothermal amplification and dipstick readout, enabling sensitive/specific on-site testing. However, improvements in sensitivity and workflow complexity are needed to spur clinical adoption. We outline the mechanisms/strategies of major CRISPR-Cas systems, evaluate their on-site diagnostic capabilities, and discuss future research directions.

Exosomal miRNAs as a Promising Source of Biomarkers in Colorectal Cancer Progression.

Colorectal cancer (CRC) is the third most common type of cancer worldwide amongst males and females. CRC treatment is multidisciplinary, often including surgery, chemotherapy, and radiotherapy. Early diagnosis of CRC can lead to treatment initiation at an earlier stage. Blood biomarkers are currently used to detect CRC, but because of their low sensitivity and specificity, they are considered inadequate diagnostic tools and are used mainly for following up patients for recurrence. It is necessary to detect novel, noninvasive, specific, and sensitive biomarkers for the screening and diagnosis of CRC at earlier stages. The tumor microenvironment (TME) has an essential role in tumorigenesis; for example, extracellular vesicles (EVs) such as exosomes can play a crucial role in communication between cancer cells and different components of TME, thereby inducing tumor progression. The importance of miRNAs that are sorted into exosomes has recently attracted scientists' attention. Some unique sequences of miRNAs are favorably packaged into exosomes, and it has been illustrated that particular miRNAs can be directed into exosomes by special mechanisms that occur inside the cells. This review illustrates and discusses the sorted and transported exosomal miRNAs in the CRC microenvironment and their impact on CRC progression as well as their potential use as biomarkers.

The Human Gut Microbiome as a Potential Factor in Autism Spectrum Disorder.

The high prevalence of gastrointestinal (GI) disorders among autism spectrum disorder (ASD) patients has prompted scientists to look into the gut microbiota as a putative trigger in ASD pathogenesis. Thus, many studies have linked the gut microbial dysbiosis that is frequently observed in ASD patients with the modulation of brain function and social behavior, but little is known about this connection and its contribution to the etiology of ASD. This present review highlights the potential role of the microbiota-gut-brain axis in autism. In particular, it focuses on how gut microbiota dysbiosis may impact gut permeability, immune function, and the microbial metabolites in autistic people. We further discuss recent findings supporting the possible role of the gut microbiome in initiating epigenetic modifications and consider the potential role of this pathway in influencing the severity of ASD. Lastly, we summarize recent updates in microbiota-targeted therapies such as probiotics, prebiotics, dietary supplements, fecal microbiota transplantation, and microbiota transfer therapy. The findings of this paper reveal new insights into possible therapeutic interventions that may be used to reduce and cure ASD-related symptoms. However, well-designed research studies using large sample sizes are still required in this area of study.

The composition and stability of the vaginal microbiome of healthy women.

Our knowledge of the composition of the vaginal environment in healthy women stands greatly improved. An imbalance in microbial communities is associated with a number of different diseases, disorders and other adverse health outcomes. Cultivation-independent studies have been published indicating that each woman has unique vaginal microbiota. The vaginal microbiome in pregnant women is more stable and associated with high level of Lactobacillus, particularly, Lactobacillus crispatus and low bacterial diversity. The current review was planned to provide a more complete picture of the abundance of various bacteria species in the vagina and how they impact women's reproductive health and pregnancy outcomes. This should provide a better understanding of what is considered a "healthy" or "unhealthy" vaginal microbiome and how the dysibiosis of the vagina affects the women. Additionally, it was planned to identify factors that influence the structure and / or composition of the microbial community.

Transcriptional analysis of Rhazya stricta in response to jasmonic acid

BACKGROUND: Jasmonic acid (JA) is a signal transducer molecule that plays an important role in plant development and stress response; it can also efficiently stimulate secondary metabolism in plant cells. RESULTS: RNA-Seq technology was applied to identify differentially expressed genes and study the time course of gene expression in Rhazya stricta in response to JA. Of more than 288 million total reads, approximately 27% were mapped to genes in the reference genome. Genes involved during the secondary metabolite pathways were up- or downregulated when treated with JA in R. stricta. Functional annotation and pathway analysis of all up- and downregulated genes identified many biological processes and molecular functions. Jasmonic acid biosynthetic, cell wall organization, and chlorophyll metabolic processes were upregulated at days 2, 6, and 12, respectively. Similarly, the molecular functions of calcium-transporting ATPase activity, ADP binding, and protein kinase activity were also upregulated at days 2, 6, and 12, respectively. Time-dependent transcriptional gene expression analysis showed that JA can induce signaling in the phenylpropanoid and aromatic acid pathways. These pathways are responsible for the production of secondary metabolites, which are essential for the development and environmental defense mechanism of R. stricta during stress conditions. CONCLUSIONS: Our results suggested that genes involved in flavonoid biosynthesis and aromatic acid synthesis pathways were upregulated during JA stress. However, monoterpenoid indole alkaloid (MIA) was unaffected by JA treatment. Hence, we can postulate that JA plays an important role in R. stricta during plant development and environmental stress conditions.

Dysbiosis of gut microbiota in inflammatory bowel disease: Current therapies and potential for microbiota-modulating therapeutic approaches.

There is a growing body of evidence reinforcing the unique connections between the host microbiome, health, and diseases. Due to the extreme importance of the symbiotic relationship between the intestinal microbiome and the host, it is not surprising that any alteration in the gut microbiota would result in various diseases, including inflammatory bowel disease (IBD), Crohn's disease, (CD) and ulcerative colitis (UC). IBD is a chronic, relapsing-remitting condition that is associated with significant morbidity, mortality, compromised quality of life, and costly medical care. Dysbiosis is believed to exacerbate the progression of IBD. One of the currently used treatments for IBD are anti-tumor necrosis factor (TNF) drugs, representing a biologic therapy that is reported to have an impact on the gut microbiota composition. The efficacy of anti-TNF agents is hindered by the possibility of non-response, which occurs in 10-20% of treated patients, and secondary loss of response, which occurs in up to 30% of treated patients. This underscores the need for novel therapies and studies that evaluate the role of the gut microbiota in these conditions. The success of any therapeutic strategy for IBD depends on our understanding of the interactions that occur between the gut microbiota and the host. In this review, the health and disease IBD-associated microbiota patterns will be discussed, in addition to the effect of currently used therapies for IBD on the gut microbiota composition, as well as new therapeutic approaches that can be used to overcome the current treatment constraints.

Identification of Causative Variants Contributing to Nonsyndromic Orofacial Clefts Using Whole-Exome Sequencing in a Saudi Family.

Objectives: Nonsyndromic orofacial clefts (NSOFCs) are the most common craniofacial malformations observed across the globe. They are classified into three types: (a) cleft palate, (b) cleft lip, and (c) cleft lip and palate. To identify the potential candidate genes contributing to polygenic diseases such as NSOFC, linkage analyses, genome-wide association studies, and genomic rearrangements can be used. Genomic analyses, based on massively parallel next-generation sequencing technologies, play a vital role in deciphering the genetic bases of NSOFCs. Materials and Methods: In this study, whole exome sequencing was employed to detect genes that likely contributed to the NSOFC phenotype in a consanguineous Saudi family. Results: The exome analysis revealed NRP1 (rs35320960) as one potential candidate gene that is involved in bone differentiation. The RPL27A gene (rs199996172), which plays a crucial role in ribosome biogenesis, also passed all filters to serve as a candidate gene for NSOFC in this family. Rare variants are situated within the 5' UTR of these two genes. Conclusion: The study suggests that rare variants in NRP1 and RPL27A may be associated with NSOFC disease etiology.

Molecular cloning, expression, overproduction and characterization of human TRAIP Leucine zipper protein.

The TRAIP interacting protein is known as a negative regulator of TNF-induced-nuclear factor, kappa-light-chain-enhancer of activated B cell (NF-κB) by direct interaction with the adaptor protein TRAF2, which inhibits the function of TRAF2 via the RINGCC domain protein. The TRAIP protein is composed of 469 amino acids with an N-terminal RING motif that is followed by a coiled coil (CC) and leucine zipper domain. TRAIP proteins are critical in programmed cell death, cell proliferation and differentiation, and embryonic development. The critical functions of TRAIP together with the molecular inhibitory mechanism effect of TRAIP have been reported by two different studies and have opened up new research into the field of TRAF biology. In this study, we designed different constructs of the Leucine zipper domain to find the over -expressed construct for further studies. We successfully cloned the C-terminal TRAIP containing the leucine zipper domain. In addition, we have over-expressed and purified the TRAIP LZ for their biochemical characterization.

Molecular evolution of cytochrome C oxidase-I protein of insects living in Saudi Arabia.

The study underpins barcode characterization of insect species collected from Saudi Arabia and explored functional constraints during evolution at the DNA and protein levels to expect the possible mechanisms of protein evolution in insects. Codon structure designated AT-biased insect barcode of the cytochrome C oxidase I (COI). In addition, the predicted 3D structure of COI protein indicated tyrosine in close proximity with the heme ligand, depicted substitution to phenylalanine in two Hymenopteran species. This change resulted in the loss of chemical bonding with the heme ligand. The estimated nucleotide substitution matrices in insect COI barcode generally showed a higher probability of transversion compared with the transition. Computations of codon-by-codon nonsynonymous substitutions in Hymenopteran and Hemipteran species indicated that almost half of the codons are under positive evolution. Nevertheless, codons of COI barcode of Coleoptera, Lepidoptera and Diptera are mostly under purifying selection. The results reinforce that codons in helices 2, 5 and 6 and those in loops 2-3 and 5-6 are mostly conserved and approach strong purifying selection. The overall results argue the possible evolutionary position of Hymenopteran species among those of other insects.

Thermopriming reprograms metabolic homeostasis to confer heat tolerance.

Heat stress threatens agriculture worldwide. Plants acquire heat stress tolerance through priming, which establishes stress memory during mild or severe transient heat stress. Such induced thermotolerance restructures metabolic networks and helps maintain metabolic homeostasis under heat stress. Here, we used an electrospray ionization mass spectrometry-based platform to explore the composition and dynamics of the metabolome of Arabidopsis thaliana under heat stress and identify metabolites involved in thermopriming. Primed plants performed better than non-primed plants under severe heat stress due to altered energy pathways and increased production of branched-chain amino acids, raffinose family oligosaccharides (RFOs), lipolysis products, and tocopherols. These metabolites serve as osmolytes, antioxidants and growth precursors to help plants recover from heat stress, while lipid metabolites help protect membranes against heat stress. The carbohydrate (e.g., sucrose and RFOs) and lipid superpathway metabolites showed the most significant increases. Under heat stress, there appears to be crosstalk between carbohydrate metabolism (i.e., the thermomemory metabolites stachyose, galactinol, and raffinose) and tyrosine metabolism towards the production of the thermomemory metabolite salidroside, a phenylethanoid glycoside. Crosstalk occurs between two glycerophospholipid pathways (the biosynthetic pathways of the thermomemory metabolite S-adenosyl-L-homocysteine and the terpenoid backbone) and the δ-tocopherol (chloroplast lipid) pathway, which favors the production of glycine betaine and other essential tocopherols, respectively, compounds which are essential for abiotic stress tolerance in plants. Therefore, metabolomic analysis can provide comprehensive insights into the metabolites involved in stress responses, which could facilitate plant breeding to maximize crop yields under adverse conditions.

Control of ß-sitosterol biosynthesis under light and watering in desert plant Calotropis procera.

Most scientific studies on Calotropis procera refer to the plant as an important source of pharmaceutical compounds and its valuable benefits in medicine. One of the most important substances in this plant is the potential immunostimulant ß-sitosterol (BS) that acts in improving human health. This study focused on the effects of lighting before and after irrigation on the BS accumulation pathway namely steroid biosynthesis. Studying the enzymes in BS biosynthetic pathway indicated the upregulation at dawn and predusk of the SMT2 and SMO2 genes encoding sterol methyltransferase 2 and methylsterol monooxygenase, two key enzymes in BS accumulation in C. procera. The results almost indicated no regulation at the different time points of the CYP710A gene encoding sterol 22-desaturase, an enzyme that acts in depleting ß-sitosterol towards the biosynthesis of stigmasterol. RNA-Seq data was validated via quantitative RT-PCR and results were positive. The data of ultra-performance liquid chromatography-tandem mass spectrometry analysis with regard to BS accumulation also aligned with those of RNA-Seq analysis. We focused on the effects of light before and after watering on BS accumulation in C. procera. Our results show that BS accumulation is high at dawn in both dehydrated and well-watered condition. While, the BS was dramatically decrease at midday in well-watered plants. This increase/decrease in BS content is correlated with rates of expression of SMT 2 gene. This gene is a key convertor between the different branches in the cardiac glycoside biosynthesis. Accordingly, it could be suggested that BS (or one of the descendent product) may play an important role in C. procera tolerance to drought/light intensity conditions.

Detection of Secondary Metabolites as Biomarkers for the Early Diagnosis and Prevention of Type 2 Diabetes.

BACKGROUND: Type 2 diabetes, or T2D, is a metabolic disease that results in insulin resistance. In the present study, we hypothesize that metabolomic analysis in blood samples of T2D patients sharing the same ethnic background can recover new metabolic biomarkers and pathways that elucidate early diagnosis and predict the incidence of T2D. METHODS: The study included 34 T2D patients and 33 healthy volunteers recruited between the years 2012 and 2013; the secondary metabolites were extracted from blood samples and analyzed using HPLC. RESULTS: Principal coordinate analysis and hierarchical clustering patterns for the uncharacterized negatively and positively charged metabolites indicated that samples from healthy individuals and T2D patients were largely separated with only a few exceptions. The inspection of the top 10% secondary metabolites indicated an increase in fucose, tryptophan and choline levels in the T2D patients, while there was a reduction in carnitine, homoserine, allothreonine, serine and betaine as compared to healthy individuals. These metabolites participate mainly in three cross-talking pathways, namely "glucagon signaling", "glycine, serine and threonine" and "bile secretion". Reduced level of carnitine in T2D patients is known to participate in the impaired insulin-stimulated glucose utilization, while reduced betaine level in T2D patients is known as a common feature of this metabolic syndrome and can result in the reduced glycine production and the occurrence of insulin resistance. However, reduced levels of serine, homoserine and allothrionine, substrates for glycine production, indicate the depletion of glycine, thus possibly impair insulin sensitivity in T2D patients of the present study. CONCLUSION: We introduce serine, homoserine and allothrionine as new potential biomarkers of T2D.

Stepwise response of MeJA-induced genes and pathways in leaves of C. roseus.

Catharanthus roseus is a perennial herb known for the production of important terpenoid indole alkaloids (TIAs) in addition to a variety of phenolic compounds. The goal of the present work was to detect the prolonged effects of MeJA (6 uM) treatment across time (up to 24 days) in order to detect the stepwise response of MeJA-induced genes and pathways in leaves of C. rouses. Prolonged exposure of plants to MeJA (6 uM) treatment for different time points (6, 12 and 24 days) indicated that genes in the indole alkaloid biosynthesis pathway and upstream pathways were triggered earlier (e.g., 6 days) than those in the anthocyanin biosynthesis pathway and its upstream pathways (e.g., 12 days). Three enzymes, e.g., T16H, OMT, and D4H, in the six-step vindoline biosynthesis and two enzymes, e.g., TDC and STR, acting consecutively in the conversion of tryptophan to strictosidine, were activated after 6 days of MeJA treatment. Two other key enzymes, e.g., TRP and CYP72A1, acting concurrently upstream of the TIA biosynthesis pathway were upregulated after 6 days. The genes encoding TDC and STR might concurrently act as a master switch of the TIA pathway towards the production of the indole alkaloids. On the other hand, we speculate that the gene encoding PAL enzyme also acts as the master switch of phenylpropanoid biosynthesis and the downstream flavonoid biosynthesis and anthocyanin biosynthesis pathways towards the production of several phenolic compounds. PAL and the downstream enzymes were activated 12 days after treatment. Cluster analysis confirmed the concordant activities of the flower- and silique-specific bHLH25 transcription factor and the key enzyme in the TIA biosynthesis pathway, e.g., STR. Due to the stepwise response of the two sets of pathways, we speculate that enzymes activated earlier likely make TIA biosynthesis pathway a more favourable target in C. roseus than anthocyanin biosynthesis pathway.