Changes in Physiological Traits, Gene Expression and Phytochemical Profile of Mentha piperita in Response to Elicitor.

Peppermint (Mentha piperita) is a perennial medicinal plant containing active ingredients that can be used for treating liver and prostate cancers, acute respiratory infections, allergies, digestive problems, neuralgia, and migraines. The objective of this research is to investigate the expression of essential genes in the menthol pathway of Mentha piperita, including Pulegone reductase (Pr), Menthofuran synthase (Mfs), and limonene synthase (Ls) using qPCR, physiological analysis and essential oil composition in response to methyl jasmonate (MeJA) (0.5 mM) elicitation. Physiological analysis showed that 0.5 mM MeJA triggers defensive responsiveness in Mentha piperita by increasing superoxide dismutase (SOD) and Peroxidase (POD) enzymes activity. The highest transcript levels of Pr and Mfs genes were observed during 8 and 12 h after treatment respectively, but following 24 h, they were down-regulated. Essential oil analysis indicated that the percentage of constituents in the essential oil was changed using MeJA at 48 h and 96 h after post-treatment. Effective antimicrobial compounds, α-pinene, ß-pinene, linalool and methyl acetate, were induced after 48 h. A non-significant positive relationship was detected between menthol content, and expression of the Pr and Mfs genes. Due to the significant change in the expression of Pr and Mfs genes in the menthol pathway, role of Pr gene in directing the pathway to the valuable compound menthol and deviation of the menthol pathway to the menthofuran as an undesirable component of essential oil by Mfs gene, it can be deduced that they are the most critical genes in response to MeJA treatment, which are appropriate candidates for metabolite engineering. In addition, MeJA improved defensive responsiveness and percentage of some constituents with antimicrobial properties in Mentha piperita.

Mixed-Ploidy and Dysploidy in Hypericum perforatum: A Karyomorphological and Genome Size Study.

Karyomorphology and genome size of 15 St John's wort (Hypericum perforatum L.) populations are reported for the first time. Root tips and fresh young leaves were used for karyological studies and flow cytometric (FCM) measurements, respectively. The chromosome length varied from 0.81 µm to 1.16 µm, and chromosome types were determined as "m". Eight different somatic chromosome numbers were found (2n = 16, 22, 24, 26, 28, 30, 32, 38). Based on the observed basic (x) chromosome numbers of x = 8, 11, 13, 14, 15, 19, this may correspond to diploid (2x), triploid (3x), tetraploid (4x), respectively. Interestingly, we found mixoploidy (3x - 4x) in the root tips of one of the populations. Hybridization, polyploidy and dysploid variation may be the main factors associated with the chromosome number evolution of this species. FCM showed that 2C DNA contents vary from 0.87 to 2.02 pg, showing more than a 2-fold variation. The mean amount of 2C DNA/chromosome and the mean of monoploid genome size were not proportional to ploidy.

Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl.

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. The effects of induced polyploidy as well as the effect of methyl Jasmonate (MeJA) elicitation on the root production of thebaine and on the expression of five alkaloid biosynthesis related genes were studied. The in vitro tetraploidy induction caused a significant increased expression of norcoclaurine synthase (NCS) and salutaridinol (SAT), and a significant decreased expression of berberine bridge enzyme (BBE) in the leaves. In the root tissues, the BBE, NCS, and SAT showed an increased expression in tetraploid plants, while codeinone reductase (COR) showed a decreased expression. A similar alteration pattern was found in mixoploid plants when compared to their diploid counterparts. MeJA at concentrations of 0.1 and 0.5 mM caused a remarkable increase in the thebaine content in the roots of treated plants, where the highest thebaine content was identified in plants elicited with 0.5 mM MeJA. Elicitation treatment caused a substantial increase in the expression of NCS and SAT in the leaves, while it had no major effect on BBE, codeine 3-O-demethylase (CODM) and COR. Expression analysis in the roots showed that MeJA caused a significant increase in the expression of only BBE and NCS, while expression of other studied genes remained unchanged. Our results may be exploited for improved thebaine production and the processing of Persian poppy.

Identification and expression analysis of S-alk(en)yl-L-cysteine sulfoxide lyase isoform genes and determination of allicin contents in Allium species.

Alliinase is the key enzyme in allicin biosynthesis pathway. In the current study, the identification and sequencing of alliinase genes along with determination of allicin contents were reported for Allium species with a novel report for Iranian endemic species. The presence of different isoforms in the Allium being discovered for the first time. In bulbs tissue, the highest allicin concentration was in Allium sativum, A. umbilicatum, and A. fistolosum (1.185%, 0.367%, and 0.34%, respectively), followed by A. spititatum (0.072%), A. lenkoranicum (0.055%), A. atroviolaseum (0.36%), A. rubellum (0.041%), and A. stamineum (0.007%). The highest allicin content in the leaves and roots were in A. sativum (0.13%), and A. stamineum (0.195%), respectively. The ORFs length ranged from 1416 in A. sativum (iso-alliinase2; ISA2) to 1523 bp in A. sativum (alliinase); the identity with A. sativum (alliinase) varies from 95% to 68% for A. ampeloprasum, and A. sativum (iso-alliinase1, ISA1) respectively. These data suggested that both ISA1 and ISA2 had a high expression in the roots and bulbs compared to A. sativum as the control in all species. Note that ISA1 and ISA2 were not expressed in the leaves. The results showed that isoforms expression patterns among different tissues in Allium species were variable. The presence of various isoforms is a possible explanation for the difference between the species in terms of obtained results, especially the amount of allicin.

Identification and Functional Analysis of Two Purple Acid Phosphatases AtPAP17 and AtPAP26 Involved in Salt Tolerance in Arabidopsis thaliana Plant.

Tolerance to salinity is a complex genetic trait including numerous physiological processes, such as metabolic pathways and gene networks; thereby, identification of genes indirectly affecting, as well as those directly influencing, is of utmost importance. In this study, we identified and elucidated the functional characterization of AtPAP17 and AtPAP26 genes, as two novel purple acid phosphatases associated with high-salt tolerance in NaCl-stressed conditions. Here, the overexpression of both genes enhanced the expression level of AtSOS1, AtSOS2, AtSOS3, AtHKT1, AtVPV1, and AtNHX1 genes, involving in the K+/Na+ homeostasis pathway. The improved expression of the genes led to facilitating intracellular Na+ homeostasis and decreasing the ion-specific damages occurred in overexpressed genotypes (OEs). An increase in potassium content and K+/Na+ ratio was observed in OE17 and OE26 genotypes as well; however, lower content of sodium accumulated in these plants at 150 mM NaCl. The overexpression of these two genes resulted in the upregulation of the activity of the catalase, guaiacol peroxidase, and ascorbate peroxidase. Consequently, the overexpressed plants showed the lower levels of hydrogen peroxide where the lowest amount of lipid peroxidation occurred in these lines. Besides the oxidation resistance, the boost of the osmotic regulation through the increased proline and glycine-betaine coupled with a higher content of pigments and carbohydrates resulted in significantly enhancing biomass production and yield in the OEs under 150 mM NaCl. High-salt stress was also responsible for a sharp induction on the expression of both PAP17 and PAP26 genes. Our results support the hypothesis that these two phosphatases are involved in plant responses to salt stress by APase activity and/or non-APase activity thereof. The overexpression of PAP17 and PAP26 could result in increasing the intracellular APase activity in both OEs, which exhibited significant increases in the total phosphate and free Pi content compared to the wild-type plants. Opposite results witnessed in mutant genotypes (Mu17, Mu26, and DM), associating with the loss of AtPAP17 and AtPAP26 functions, clearly confirmed the role of these two genes in salt tolerance. Hence, these genes can be used as candidate genes in molecular breeding approaches to improve the salinity tolerance of crop plants.

Metabolite profiling and molecular responses in a drought-tolerant savory, Satureja rechingeri exposed to water deficit.

This study aimed to determine the response of Satureja rechingeri to water deficit by quantifying the expression of three targeted genes and four traditional reference genes using quantitative real-time PCR analysis (RT-qPCR). Drought stress was imposed by withholding water 4 months after planting. Profiling of volatile and non-volatile compounds using gas chromatography/mass spectrometry (GC/MS) and high-performance thin layer chromatography (HPTLC) showed an increasing-decreasing trend of major phenolic and terpenoid compounds such as rosmarinic and caffeic acids, carvacrole, thymol and p-Cymene. Drought stress also lead to significant increases in oil yield, soluble sugars and proline as well as significant reductions in leaf water potential (LWP), relative water content (RWC), and pigments. Metabolite profiling revealed the strategies savory employed to generate different biochemical phenotypes. RT-qPCR analysis showed that up-regulation of the three genes [1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 3-hydroxy-3-methylglutaryl-coenzyme. A reductase (HMGR) and rosmarinic acid synthase: 4-coumaroyl-CoA (RAS)] selected from the phenylpropanoid and terpenoid biosynthesis pathways were markedly enhanced at the transcript levels of the regulatory steps and directly increased the production of secondary metabolites, including phenolic and terpenoid compounds. Actin protein (ACT), elongation factor 1-α (EF1α), glyceraldehyde-3-phosphate dehydrogenase cytosolic (GAPC) and ubiquitin-conjugating enzyme (UBC) were used as traditional reference genes. UBC's suitability as the reference genes were verified in S. rechingeri. The study's results provide the foundation for gene expression analysis of savory and other species of Lamiaceae. Thus, the effective application of drought stress before harvesting can increase the quantity and quality of raw material.

Author Correction: Expression of key genes affecting artemisinin content in five Artemisia species.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Expression of key genes affecting artemisinin content in five Artemisia species.

Artemisinin, an effective anti-malarial drug is synthesized in the specialized 10-celled biseriate glandular trichomes of some Artemisia species. In order to have an insight into artemisinin biosynthesis in species other than A. annua, five species with different artemisinin contents were investigated for the expression of key genes that influence artemisinin content. The least relative expression of the examined terpene synthase genes accompanied with very low glandular trichome density (4 No. mm-2) and absence of artemisinin content in A. khorassanica (S2) underscored the vast metabolic capacity of glandular trichomes. A. deserti (S4) with artemisinin content of 5.13 mg g-1 DW had a very high expression of Aa-ALDH1 and Aa-CYP71AV1 and low expression of Aa-DBR2. It is possible to develop plants with high artemisinin synthesis ability by downregulating Aa-ORA in S4, which may result in the reduction of Aa-ALDH1 and Aa-CYP71AV1 genes expression and effectively change the metabolic flux to favor more of artemisinin production than artemisinic acid. Based on the results, the Aa-ABCG6 transporter may be involved in trichome development. S4 had high transcript levels and larger glandular trichomes (3.46 fold) than A. annua found in Iran (S1), which may be due to the presence of more 2C-DNA (3.48 fold) in S4 than S1.

Assessment of Iranian endemic Artemisia khorassanica: karyological, genome size, and gene expressions involved in artemisinin production.

The species of Artemisia, one of the largest genera of the family Asteraceae, are frequently utilized for the treatment of diseases such as malaria, hepatitis, cancer, inflammation, and infections by fungi, bacteria, and viruses. Karyological studies were performed on 18 Artemisia khorassanica populations: eleven were diploid (2n = 18) and seven were tetraploid (2n = 36). The mean chromosome lengths were 3.61 and 3.84 µm for diploids and tetraploids, respectively. Two chromosome types ("m", "sm") formed karyotype formulas "18m" for diploids and "36m" and "34m + 2sm" for tetraploids. The mean 2C DNA contents were 5.91 and 11.53 pg in diploids and tetraploids, respectively. The transcription levels of key genes involved in artemisinin production were compared in diploid (B, D, H) and tetraploid (O, P, R) A. khorassanica relative to A. annua as a standard species. No artemisinin content was detected in diploid and tetraploid A. khorassanica populations. No significant diefrences were detected between diploids and tetraploids in terms of DXR , HMGR, FDS, and ADS gene expression. This implies that most of the genomic amplification likely occurs in the amount of repetitive DNA and not in unique sequences. The DBR2 gene was expressed in the diploid A. khorassanica in a low amount but silenced in the autotetraploid A. khorassanica.

In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

MAIN CONCLUSION: Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72 h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.

Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L.

Papaver somniferum L. is an herbaceous, annual and diploid plant that is important from pharmacological and strategic point of view. The cDNA clones of two putative MYB and WRKY genes were isolated (GeneBank accession numbers KP411870 and KP203854, respectively) from this plant, via the nested-PCR method, and characterized. The MYB transcription factor (TF) comprises 342 amino acids, and exhibits the structural features of the R2R3MYB protein family. The WRKY TF, a 326 amino acid-long polypeptide, falls structurally into the group II of WRKY protein family. Quantitative real-time PCR (qRT-PCR) analyses indicate the presence of these TFs in all organs of P. somniferum L. and Papaver bracteatum L. Highest expression levels of these two TFs were observed in the leaf tissues of P. somniferum L. while in P. bracteatum L. the espression levels were highest in the root tissues. Promoter analysis of the 10 co-expressed gene clustered involved in noscapine biosynthesis pathway in P. somniferum L. suggested that not only these 10 genes are co-expressed, but also share common regulatory motifs and TFs including MYB and WRKY TFs, and that may explain their common regulation.

Proteomic analysis of a spring wheat cultivar in response to prolonged cold stress.

Cold represents one of the major abiotic factors influencing plant growth and development worldwide. We analysed the long-term responsiveness of an Iranian spring wheat (cv. Kohdasht) to cold from a proteomic point of view, in order to unravel the molecular mechanisms helping a cold-sensitive cultivar to survive exposure to suboptimal temperatures. Plants were grown at 20 or 4°C until entering the reproductive stage and a cross-comparison on the leaf proteomes was performed. Quantitative analyses on protein alterations occurring upon low-temperature exposure showed a reinforcement in ascorbate recycling (dehydroascorbate reductase, ascorbate peroxidase) and protein processing (proteasome subunit, cysteine proteinase), as well as the accumulation of the enzyme devoted to tetrapyrrole resynthesis (glutamate semialdehyde aminomutase). In contrast, among proteins down-regulated after cold stress, we could identify some key Krebs cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase), together with many photosynthesis-related proteins (oxygen-evolving complex proteins, ATP synthase subunits, ferredoxin NADPH oxidoreductase and some Calvin cycle enzymes). Physiological and biochemical parameters (such as shoot apex dissection, chlorophyll, proline and sugar content determination) sustained proteomics findings allowing the present research to contribute to the current knowledge on these long-term responses, which may be crucial to stress adaptation under field conditions.

Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.).

Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-ß-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur.

Abscisic acid regulates gene expression in cortical fiber cells and silica cells of rice shoots.

Drought-induced growth arrest is a major cause of yield loss in crops and is mediated in part by abscisic acid (ABA). The aim of this study was to identify the cell types targeted by ABA during arrest. As transcription factors ABI3 and ABI5 are essential for ABA-induced growth arrest in Arabidopsis, blast was used to identify OsVP1 and OsABF1 as their structural orthologues in rice (Oryza sativa), and employed RNA in situ hybridization to reveal the cell types accumulating the corresponding transcripts in response to ABA. Exogenous ABA arrested the growth of leaves 1, 2 and 3 in young rice shoots and inhibited secondary cell-wall formation in sclerenchyma, including expression of the cellulose synthase gene OsCesA9. Transcripts for OsVP1, OsABF1 and of the putative target genes OsEm, OsLEA3 and WSI18, increased under ABA, accumulating principally in the cytosol of the major support cells (sclerenchymatous cortical fiber cells and epidermal silica cells) of slowly growing leaf 1. Rapidly growing immature tissues in leaves 2 and 3 accumulated OsABF1, OsEm and WSI18 transcripts in the nuclei of all cells, irrespective of ABA treatment. It is concluded that during arrest of leaf growth, ABA targets support cells in maturing tissues. Target cells in immature tissues remain to be identified.