Change in Secondary Metabolites and Expression Pattern of Key Rosmarinic Acid Related Genes in Iranian Lemon Balm (Melissa officinalis L.) Ecotypes Using Methyl Jasmonate Treatments.

The medicinal herb, lemon balm (Melissa officinalis L.), which is high in rosmarinic acid (RA), has well-known therapeutic value. The goals of this study were to investigate the effects of methyl jasmonate (MeJA) on RA content, total phenolic content (TPC), and total flavonoid content (TFC), as well as changes in expression of their biosynthesis-related key genes (MoPAL, Mo4CL, and MoRAS) in Iranian lemon balm ecotypes, as first reported. Our results revealed that MeJA doses significantly increase the RA content, TPC, and TFC in both ecotypes compared with the control samples. Additionally, the higher expression levels of MoPAL, Mo4CL, and MoRAS following treatment were linked to RA accumulation in all treatments for both Iranian lemon balm ecotypes. After 24 h of exposure to 150 µM MeJA concentration, HPLC analysis showed that MeJA significantly increased RA content in Esfahan and Ilam ecotypes, which was about 4.18- and 7.43-fold higher than untreated plants. Our findings suggested that MeJA has a considerable influence on RA, TPC, and TFC accumulation in MeJA-treated Iranian M. officinalis, which might be the result of gene activation from the phenylpropanoid pathway. As a result of our findings, we now have a better understanding of the molecular processes behind RA production in lemon balm plants.

Effect of Methyl Jasmonate on Thymol, Carvacrol, Phytochemical Accumulation, and Expression of Key Genes Involved in Thymol/Carvacrol Biosynthetic Pathway in Some Iranian Thyme Species.

Thyme species are a good source of thymol and carvacrol, which play a key role in controlling diseases. For the first time, the expression patterns of γ-terpinene synthase (TPS2), CYP71D178, and CYP71D180 genes and the amount of phenolics compounds were evaluated in T. migricus and T. daenensis after different methyl jasmonate (MeJA) treatments. The highest thymol and carvacrol contents were observed in T. migricus (86.27%) and T. daenensis (17.87%) at MeJA 100 µM, which was consistent with the expression patterns of the three investigated genes. All species treated showed high total phenolic and flavonoid content compared to control plants for which the highest amounts were observed in T. vulgaris treated with 100 µM and 10 µM MeJA. Furthermore, in the 100 µM MeJA treatment, the relative expression of TPS2 and CYP71D178 in T. migricus increased 7.47 and 9.86-fold compared with the control, respectively. The highest level of CYP71D180 transcripts (5.15-fold) was also observed for T. daenensis treated. This finding highlights the notion that thymol was known as the dominant component of the essential oil rather than carvacrol in diffident thyme species. This implies that MeJA at different concentrations influenced metabolic pathways and induced expression changes, resulting in a rise in essential oil levels.

Ipomoea batatas: papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites.

Different parasites cause severe lose in quantity and quality of crops. Many parasites develop haustorial cells and stylets that penetrate the host using secreted enzymes and mechanical pressure. Cysteine proteases are pre-pro-enzyme produced by parasites that are essential for normal parasitism. Papain is also a kind of cysteine proteases such that its propeptide segment has inhibitory properties and limits the protease activity of papain. To investigate the inhibitory effects of papain propeptide on some parasite proteases, we cloned inhibitory propeptide of papain of Ipomoea batatas, and enzymatic fragments of Diabrotica virgifera cathepsin L-like protease-1, Meloidogyne incognita cathepsin L-like protease 1, Heterodera glycines cysteine protease-1, Cuscuta chinesis cysteine protease and Orobanche cernua cysteine protease. After purification of recombinant inhibitory propeptide and enzymatic fragments, the inhibition activity of propeptide on cysteine proteases was measured. Finally inhibitory propeptide was transformed into tomato and transgenic plants resistance to parasites (bioassay) were examined. We demonstrated papain-propeptide inhibits cysteine protease of mentioned parasites. In transgenic tomato plants, papain-inhibitory propeptide effectively interrupted haustoria development. Haustoria-digitate cells of dodder could not differentiate and develop into the phloem and xylem hyphae on transgenic tomatoes. Parasites grown on transgenic tomatoes showed reduction in vigor and productivity due to defective connection of haustoria. Lower ratio of female nematodes and a decrease of nematode egg mass per transgenic line indicated biocontrol of nematode. The changes in growth factors of parasite challenged transgenic lines relative to controls, indicates the efficacy of papain propeptide in control of parasitism.

Histochemical Analysis and Ultrastructure of Trichomes and Laticifers of Croton gratissimus Burch. var. gratissimus (Euphorbiaceae).

Croton gratissimus (Lavender croton) possesses three distinct secretory structures. These include lepidote and glandular trichomes and non-articulated unbranched laticifers. The lepidote trichomes form a dense indumentum on the abaxial surface of the leaves and canopy the glandular trichomes. Although assumed to be non-glandular, transmission electron microscopy (TEM) indicated high metabolic activity within the stalk and radial cells. Glandular trichomes are embedded in the epidermal layer and consist of a single cell which forms a prominent stalk and dilated head. Laticifers occur on the mid-vein of leaves and are predominantly associated with vascular tissue. In the stems, laticifers are associated with the phloem and pith. Both trichome types and laticifers stained positive for alkaloids, phenolic compounds, and lipids. Positive staining for these compounds in lepidote trichomes suggests their involvement in the production and accumulation of secondary metabolites. These metabolites could provide chemical defense for the plant and potentially be useful for traditional medicine.

Antioxidant and Antibacterial Activities of the Leaf and Stem Extracts of Combretum molle (R. Br. ex G. Don.) Engl. & Diels.

Medicinal plants offer reasonable and accessible alternatives to synthetic drugs and are often devoid of the adverse side effects, toxicity, and pathogenic resistance associated with synthetic medicine. Combretum molle has been utilized in African traditional medicinal practices and purportedly contains bioactive compounds with medicinally beneficial effects. This study investigated the hexane, chloroform, and methanol leaf and stem extracts for their antioxidant properties using the 2,2'-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing antioxidant power assays. The study additionally analyzed the methanol extracts for their antibacterial activity against Gram-negative Escherichia coli (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 25923) bacteria using agar well diffusion. Relative to the scavenging activity of the ascorbic acid control (79.15 ± 0.63% at 15 µg/mL to 94.61 ± 0.12% at 240 µg/mL), the plant's radical scavenging activities were exceptionally high in the methanolic leaf and stem extracts (p < 0.05), ranging from 94.58 ± 1.10% at 15 µg/mL to 99.22 ± 0.30% at 240 µg/mL and 91.57 ± 1.71% at 15 µg/mL to 99.60 ± 0.20% at 240 µg/mL, respectively, suggesting a strong capacity to donate hydrogen ions. High scavenging activities were additionally observed in the chloroform stem (78.68 ± 1.18% at 15 µg/mL to 98.14 ± 1.22% at 240 µg/mL) and hexane leaf (72.12 ± 4.38% at 15 µg/mL to 89.87 ± 1.50% at 240 µg/mL) extracts (p < 0.05). All extracts exhibited poor ferric-reducing abilities in relation to the gallic acid control (100 ± 0.00%) at all concentrations (p < 0.05). The leaf and stem extracts exhibited broad-spectrum antibiotic capabilities against both tested strains, with significant activity at higher concentrations (p < 0.05). Overall, both the leaf and stem extracts of C. molle exhibited similar antioxidant and antibacterial activities. These findings warrant further pharmacological research on C. molle for potential drug development.

The Influence of Methyl Jasmonate on Expression Patterns of Rosmarinic Acid Biosynthesis Genes, and Phenolic Compounds in Different Species of Salvia subg. Perovskia Kar L.

Salvia yangii B.T. Drew and Salvia abrotanoides Kar are two important fragrant and medicinal plants that belong to the subgenus Perovskia. These plants have therapeutic benefits due to their high rosmarinic acid (RA) content. However, the molecular mechanisms behind RA generation in two species of Salvia plants are still poorly understood. As a first report, the objectives of the present research were to determine the effects of methyl jasmonate (MeJA) on the rosmarinic acid (RA), total flavonoid and phenolic contents (TFC and TPC), and changes in the expression of key genes involved in their biosynthesis (phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and rosmarinic acid synthase (RAS)). The results of High-performance liquid chromatography (HPLC) analysis indicated that MeJA significantly increased RA content in S. yungii and S. abrotanoides species (to 82 and 67 mg/g DW, respectively) by 1.66- and 1.54-fold compared with untreated plants. After 24 h, leaves of Salvia yangii and Salvia abrotanoides species treated with 150 M MeJA had the greatest TPC and TFC (80 and 42 mg TAE/g DW, and 28.11 and 15.14 mg QUE/g DW, respectively), which was in line with the patterns of gene expression investigated. Our findings showed that MeJA dosages considerably enhanced the RA, TPC, and TFC contents in both species compared with the control treatment. Since increased numbers of transcripts for PAL, 4CL, and RAS were also detected, the effects of MeJA are probably caused by the activation of genes involved in the phenylpropanoid pathway.

An Analysis of Genetic Variability and Population Structure in Wheat Germplasm Using Microsatellite and Gene-Based Markers.

Knowledge of the natural patterns of genetic variation and their evolutionary basis is required for sustainable management and conservation of wheat germplasm. In the current study, the genetic diversity and population structure of 100 individuals from four Triticum and Aegilops species (including T. aestivum, Ae. tauschii, Ae. cylindrica, and Ae. crassa) were investigated using two gene-based markers (start codon targeted (SCoT) polymorphism and CAAT-box derived polymorphism (CBDP)) and simple-sequence repeats (SSRs). The SCoT, CBDP, and SSR markers yielded 76, 116, and 48 polymorphism fragments, respectively. The CBDP marker had greater efficiency than the SCoT and SSR markers due to its higher polymorphism content information (PIC), resolving power (Rp), and marker index (MI). Based on an analysis of molecular variance (AMOVA) performed using all marker systems and combined data, there was a higher distribution of genetic variation within species than among them. Ae. cylindrica and Ae. tauschii had the highest values for all genetic variation parameters. A cluster analysis using each marker system and combined data showed that the SSR marker had greater efficiency in grouping of tested accessions, such that the results of principal coordinate analysis (PCoA) and population structure confirmed the obtained clustering patterns. Hence, combining the SCoT and CBDP markers with polymorphic SSR markers may be useful in genetic fingerprinting and fine mapping and for association analysis in wheat and its germplasm for various agronomic traits or tolerance mechanisms to environmental stresses.

Methyl Jasmonate Induces Genes Involved in Linalool Accumulation and Increases the Content of Phenolics in Two Iranian Coriander (Coriandrum sativum L.) Ecotypes.

The medicinal herb coriander (Coriandrum sativum L.), with a high linalool (LIN) content, is widely recognized for its therapeutic benefits. As a novel report, the goals of this study were to determine how methyl jasmonate (MeJA) affects total phenolic content (TPC), LIN content, flavonoid content (TFC), and changes in gene expression involved in the linalool biosynthesis pathway (CsγTRPS and CsLINS). Our findings showed that, in comparison to the control samples, MeJA treatment substantially enhanced the TPC, LIN, and TFC content in both ecotypes. Additionally, for both Iranian coriander ecotypes, treatment-induced increases in CsγTRPS and CsLINS expression were connected to LIN accumulation in all treatments. A 24 h treatment with 150 µM MeJA substantially increased the LIN content in the Mashhad and Zanjan ecotypes, which was between 1.48 and 1.69 times greater than that in untreated plants, according to gas chromatography-mass spectrometry (GC-MS) analysis. Our findings demonstrated that MeJA significantly affects the accumulation of LIN, TPC, and TFC in Iranian C. sativum treated with MeJA, which is likely the consequence of gene activation from the monoterpene biosynthesis pathway. Our discoveries have improved the understanding of the molecular mechanisms behind LIN synthesis in coriander plants.

Applications of CRISPR-Cas9 as an Advanced Genome Editing System in Life Sciences.

Targeted nucleases are powerful genomic tools to precisely change the target genome of living cells, controlling functional genes with high exactness. The clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) genome editing system has been identified as one of the most useful biological tools in genetic engineering that is taken from adaptive immune strategies for bacteria. In recent years, this system has made significant progress and it has been widely used in genome editing to create gene knock-ins, knock-outs, and point mutations. This paper summarizes the application of this system in various biological sciences, including medicine, plant science, and animal breeding.

Identification and tissue-specific expression of rutin biosynthetic pathway genes in Capparis spinosa elicited with salicylic acid and methyl jasmonate.

Capparis spinosa is an edible medicinal plant which is considered as an excellent source of rutin. Rutin is a glycoside of the flavonoid quercetin that has been reported to have a beneficial role in controlling various diseases such as hypertension, arteriosclerosis, diabetes, and obesity. In this study, the partial cDNA of four genes involved in the rutin biosynthetic pathway including 4-coumaroyl CoA ligase (4CL), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) and flavonol-3-O-glucoside L-rhamnosyltransferase (RT) were identified in C.spinosa plants for the first time. The protein sequences of these genes shared high similarity with the same proteins in other plant species. Subsequently, the expression patterns of these genes as well as rutin accumulation in C.spinosa leaves treated with different concentrations of salicylic acid (SA) and methyl jasmonate (MeJA) and also in different tissues of Caper plants treated with 100 mgL-1 SA and 150 µM MeJA were evaluated. The expression of all four genes was clearly up-regulated and rutin contents increased in response to MeJA and SA treatments after 24 h. The highest rutin contents (5.30 mgg-1 DW and 13.27 mgg-1 DW), as well as the highest expression levels of all four genes, were obtained using 100 mgL-1 SA and 150 µM MeJA, respectively. Among the different tissues, the highest rutin content was observed in young leaves treated with 150 µM MeJA, which corresponded to the expression of related genes, especially RT, as a key gene in the rutin biosynthetic pathway. These results suggest that rutin content in various tissues of C. spinosa can be enhanced to a significant extent by MeJA and SA treatments and the gene expression patterns of rutin-biosynthesis-related genes are regulated by these elicitors.