Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.

Evaluation of biochemical, physiological traits and percentage of essential oil of sickleweed (Falcaria vulgaris) population in different geographical and climatic regions.

Sickleweed (Falcaria vulgaris) is the name of a species of annual, and perennial herbaceous plants of the genus Falcaria. Climate change could negatively influnces the performance of various plant species in plant kingdom. In this study, 15 different sickleweed populations from seven provinces of the country were collected based on an unbalanced nest design with 10 replications and the percentage of essential oil, types of chlorophyll, phenol, proline, protein, and carotenoids were measured on them. The results showed that there was a significant difference between populations at the level of one percent for the studied traits. The results of the mean comparison showed that populations Ard-Shaban and Qaz-Ilan in terms of the evaluated traits and especially the percentage of essential oil were at the upper of the studied samples and selected as suitable populations. In addition, populations Gilan-Deylaman and Kur-Gerger-e Sofla were also identified as superior populations in terms of studied traits by cluster analysis and principle component analysis (PCA). Since the high level of proline and biochemical and physiological traits in plants can play a role in plants' tolerance to stresses, therefore, populations with high values of these traits can be used in stress tolerance breeding programs. Therefore, in this study, populations Gilan-Deylaman and Kur-Gerger-e Sofla can be suitable populations for this purpose. In addition, the essential oil of this plant is used in the treatment of diseases, and therefore populations Ard-Shaban and Qaz-Ilan, which showed a high percentage of essential oil, can be used in breeding programs to increase the performance of this trait.

Comparison of carbohydrate partitioning and expression patterns of some genes involved in carbohydrate biosynthesis pathways in annual and biennial species of Cichorium spp.

Variation in metabolism and partitioning of carbohydrates, particularly fructans, between annual and perennial Cichorium species remains a challenging topic. To address this problem, an annual (endive, Cichorium endive L. var. Crispum; Asteraceae) and a biennial species (chicory, Cichorium intybus L. var. Witloof; Asteraceae) were compared with in terms of variability in carbohydrate accumulation and expression patterns of fructan-active enzyme genes, as well as sucrose metabolism at various growth and developmental stages. In general, constituents such as 1-kestose, nystose, and inulin were detected only in the root of chicory and were not present in any of the endive tissues. For both species, flower tissue contained maximum levels of both fructose and glucose, while for sucrose, more fluctuations were observed. On the other hand, all the genes under study exhibited variation, not only between the two species but also among different tissues at different sampling times. In endive root compared to endive leaf, the expression of cell wall invertase genes and sucrose accumulation decreased simultaneously, indicating the limited capacity of its roots to absorb sucrose, a precursor to inulin production. In addition, low expression of fructan: fructan fructosyltransferase in endive root compared to chicory root confirmed the inability of endive to inulin synthesis. Overall, annual and biennial species were different in the production of inulin, transport, remobilization, and unloading of sucrose.

Proficient dye removal from water using biogenic silver nanoparticles prepared through solid-state synthetic route.

An environmentally benign, one-pot and highly scalable method was presented to produce biogenic silver nanoparticles (Ag NPs) using the solid-state synthetic route. Four plant-derived candidate bio-reductants (i.e., Datura stramonium, Papaver orientale, Mentha piperita, and Cannabis sativa) were investigated to compare the efficiency of solid-state route and typical solution method. M. piperita was selected as the best plant resource to produce totally pure and uniform Ag NPs (average diameter of 15 nm) without any aggregation. The purity and size of biogenic Ag NPs, were tailored by adjusting the M. piperita leaf powder/silver nitrate weight ratio and temperature. The as-synthesized Ag NPs were effectively utilized as an eco-friendly nanoadsorbent in water remediation to remove a model dye (i.e., crystal violet). The key factors affecting on the sorption process (i.e., nanoadsorbent dosage, temperature, pH, dye initial concentration, and shaking time) were investigated. The pseudo-second-order kinetic model was well fitted to the sorption process and at the optimum sorption conditions, based on the Langmuir model, the adsorption capacity was found to be 704.7 mg/g. The current, cost effective and feasible method could be considered as an applicable strategy to produce green, reusable and proficient Ag NPs as nanoadsorbents for removal of dyes from contaminated water.

Tissue-Specific Transcriptome Analysis Reveals Candidate Genes for Terpenoid and Phenylpropanoid Metabolism in the Medicinal Plant Ferula assafoetida.

Ferula assafoetida is a medicinal plant of the Apiaceae family that has traditionally been used for its therapeutic value. Particularly, terpenoid and phenylpropanoid metabolites, major components of the root-derived oleo-gum-resin, exhibit anti-inflammatory and cytotoxic activities, thus offering a resource for potential therapeutic lead compounds. However, genes and enzymes for terpenoid and coumarin-type phenylpropanoid metabolism have thus far remained uncharacterized in F. assafoetida Comparative de novo transcriptome analysis of roots, leaves, stems, and flowers was combined with computational annotation to identify candidate genes with probable roles in terpenoid and coumarin biosynthesis. Gene network analysis showed a high abundance of predicted terpenoid- and phenylpropanoid-metabolic pathway genes in flowers. These findings offer a deeper insight into natural product biosynthesis in F. assafoetida and provide genomic resources for exploiting the medicinal potential of this rare plant.

Removal of crystal violet from water using ß-cyclodextrin functionalized biogenic zero-valent iron nanoadsorbents synthesized via aqueous root extracts of Ferula persica.

Three brands of zero-valent iron nanoparticles (Fe° NPs) were biologically/chemically fabricated, and sorption capacities thereof in crystal violet (CV) water remediation were compared and contrasted. Meanwhile, the ß-Cyclodextrin (ßCD) amounts effects on the size and structure of Fe° NPs were evaluated via field emission scanning electron microscopy, elemental mapping, X-ray diffraction, transmission electron microscopy, Brunauer-Emmett-Teller, and zeta potential analyses. Separated spherical ultra-small Fe° NPs (6.1 nm) with a narrower size distribution could be produced at higher dosages of ßCD. The green synthesized Fe° NPs showed better performance than those produced chemically in CV removal (39.5% vs. 14.7%, respectively) because of their higher surface area (46.68 m2/g vs. 34.38 m2/g, respectively). ßCD functionalized Fe° NPs could double nanoadsorbent proficiency in CV removal (99.8%), possibly because of simultaneous decrement in the nanoparticles sizes and increment in the active sorption sites of nanoadsorbent. The effects of nanoadsorbent amount, pH, contact time, temperature, and initial concentration on the sorption were all scrutinized. The adsorption kinetics were found to be finely fitted with the pseudo-second-order model. Adsorption capacity calculated by Langmuir model was 454.5 mg/g (20 °C, at pH 9.0). The current green, reusable, and low-cost nanoadsorbent could be utilized proficiently for practical water remediation.

Spatiotemporal oscillations of morphinan alkaloids in opium poppy.

Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT, COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part of stem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulous bud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage, while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times. Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, while negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from upper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achieve high amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all the five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling times. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebaine biosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes of T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the producing (source) tissues to the roots (sink), known as a phenomenon of 'source-to-sink transportation'. The results, altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal oscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opium poppy.

Water deficit stress fluctuates expression profiles of 4Cl, C3H, COMT, CVOMT and EOMT genes involved in the biosynthetic pathway of volatile phenylpropanoids alongside accumulation of methylchavicol and methyleugenol in different Iranian cultivars of basil.

Here, for the first time, the accumulation ratio of methylchavicol and methyleugenoland compounds together with the expression profiles of five critical genes (i.e., 4Cl, C3H, COMT, CVOMT and EOMT) in three Iranian cultivars of basil were assessed under water deficit stress at flowering stage. The highest value of methylchavicol was detected for Cul. 3 under severe stress (S3; 7.695µg/mg) alongside Cul. 2 under similar circumstances (S3; 4.133µg/mg), while regarding Cul. 1, no detectable amounts were acquired. Considering methyleugenol, Cul. 3 (0.396µg/mg; S0) followed by Cul. 1 (S3; 0.160µg/mg) were the capable plant samples in producing some detectable amounts of methyleugenol. Apart from some expectations, all the genes under study exhibited also different transcription ratios under deficit stress. Our results, overall, demonstrated that the regulation of the above-mentioned genes and production of methychavicol and methyleugenol seems to be a cultivar- and drought stress-dependent mechanism.

Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata.

KEY MESSAGE: Environmental cues have synergistic or antagonistic regulatory roles on transcription activity and taxanes accumulation in yew, though DBAT activity is less influenced, could be accordingly a rate-limiting enzyme. The current work was undertaken to elucidate the consequences of some environmental cues (i.e., day length, temperature, sunlight and relative humidity) on the expression patterns of TXS, DBAT, BAPT and DBTNBT genes contributed to the taxol biosynthetic pathway along with the accumulation of some taxanes in needles and stems of Taxus baccata over year 2013-2014. In both tissues, light intensity and temperature correlated with the production of 10-DAB III and total taxanes, and TXS activity, while a lack of significant association was deduced for day length and relative humidity. Furthermore, in both tissues, a weak correlation was observed between BAC III and light intensity, temperature, day length and relative humidity, and the corresponding gene, DBAT. Surprisingly, DBAT activity was not co-induced with TXS in both tissues, and remained expressed at basal levels over year, supporting that the conversion of 10-DAB III into BAC III could presumably be a rate limiting step in the taxol biosynthetic pathway. Similar to BAC III, no strong correlation was detected between production of taxol in both tissues and all the meteorological data, while the corresponding genes BAPT and DBTNBT, in some cases, exhibited significant correlated results. Notably, despite higher activities of BAPT and DBTNBT in both tissues over year, taxol production was still in small quantities, probably owing to the low amounts of its precursors rather than low volumes of BAPT and DBTNBT transcripts. The results, altogether, could provide us new insights towards the potential regulatory roles of environmental cues on the production of taxanes in yew trees.

Phylogenetic analysis of PR genes in some pome fruit species with the emphasis on transcriptional analysis and ROS response under Erwinia amylovora inoculation in apple.

Attempts were made to identify eight pathogenesis related (PR) genes (i.e., PR-1a, PR3-ch1, PR3-Ch2, PR3-Ch3, PR3-Ch4, PR3-Ch5, PR-5 and PR-8) from 27 genotypes of apple, quince and pear, which are induced in response to inoculation with the pathogen Erwinia amylovora, the causal agent of fire blight. Totally, 32 PR genes of different families were obtained, excepting PR3-Ch2 (amplified only in apple) and PR3-Ch4 (amplified only in apple and pear), the others were successfully amplified in all the genotypes of apple, quince and pear. Evolutionary, the genes of each family exhibited significant homology with each other, as the corresponded phylogenetic neighbor-joining-based dendrograms were taken into consideration. Meanwhile, according to the expression assay, it was deduced that the pathogen activity can significantly affect the expression levels of some selected PR genes of PR3-Ch2, PR3-Ch4, PR3-Ch5 and particularly Cat I in both resistant (MM-111) and semi-susceptible (MM-106) apple rootstocks. Lastly, it was concluded that the pathogen E. amylovora is able to stimulate ROS response, particularly using generation of hydrogen peroxide (H2O2) in both aforementioned apple rootstock.

Precision assessment of some supervised and unsupervised algorithms for genotype discrimination in the genus Pisum using SSR molecular data.

For the first time, prediction accuracies of some supervised and unsupervised algorithms were evaluated in an SSR-based DNA fingerprinting study of a pea collection containing 20 cultivars and 57 wild samples. In general, according to the 10 attribute weighting models, the SSR alleles of PEAPHTAP-2 and PSBLOX13.2-1 were the two most important attributes to generate discrimination among eight different species and subspecies of genus Pisum. In addition, K-Medoids unsupervised clustering run on Chi squared dataset exhibited the best prediction accuracy (83.12%), while the lowest accuracy (25.97%) gained as K-Means model ran on FCdb database. Irrespective of some fluctuations, the overall accuracies of tree induction models were significantly high for many algorithms, and the attributes PSBLOX13.2-3 and PEAPHTAP could successfully detach Pisum fulvum accessions and cultivars from the others when two selected decision trees were taken into account. Meanwhile, the other used supervised algorithms exhibited overall reliable accuracies, even though in some rare cases, they gave us low amounts of accuracies. Our results, altogether, demonstrate promising applications of both supervised and unsupervised algorithms to provide suitable data mining tools regarding accurate fingerprinting of different species and subspecies of genus Pisum, as a fundamental priority task in breeding programs of the crop.

Comparative study of adsorptive role of carbonaceous materials in removal of UV-active impurities of paclitaxel extracts.

Graphite oxide (GO) and reduced graphene oxide (rGO) nanosheets were synthesized with a low-cost manufacturing method. The morphology and structures of the synthesized samples were studied using X-ray diffraction (XRD), atomic force microscopy (AFM), Fourier-transform infrared (FTIR) and Raman spectroscopy. The efficiencies of GO and rGO as novel candidate adsorbents in the pre-purification of paclitaxel were compared and contrasted with those of commercial graphite (Gt), graphene (G) and multi-wall carbon nanotube (MWCNT). According to UV-vis and HPLC analyses, rGO was evaluated as the best absorbent for the removal of impurities in pre-purification of paclitaxel from plant cell cultures. In contrast, the GO had the poorest proficiency for paclitaxel pre-purification in comparison with the other carbonaceous adsorbents. This is attributed to the existence of many localized defects in the π-structure of GO that is related to weakness of π-π stacking interactions between crude extract impurities and GO.

Gene identification programs in bread wheat: a comparison study.

Seven ab initio web-based gene prediction programs (i.e., AUGUSTUS, BGF, Fgenesh, Fgenesh+, GeneID, Genemark.hmm, and HMMgene) were assessed to compare their prediction accuracy using protein-coding sequences of bread wheat. At both nucleotide and exon levels, Fgenesh+ was deduced as the superior program and BGF followed by Fgenesh were resided in the next positions, respectively. Conversely, at gene level, Fgenesh with the value of predicting more than 75% of all the genes precisely, concluded as the best ones. It was also found out that programs such as Fgenesh+, BGF, and Fgenesh, because of harboring the highest percentage of correct predictive exons appear to be much more applicable in achieving more trustworthy results, while using both GeneID and HMMgene the percentage of false negatives would be expected to enhance. Regarding initial exon, overall, the frequency of accurate recognition of 3' boundary was significantly higher than that of 5' and the reverse was true if terminal exon is taken into account. Lastly, HMMgene and Genemark.hmm, overall, presented independent tendency against GC content, while the others appear to be slightly more sensitive if GC-poor sequences are employed. Our results, overall, exhibited that to make adequate opportunity in acquiring remarkable results, gene finders still need additional improvements.

Genetic diversity of selected Iranian quinces using SSRs from apples and pears.

An Iranian National Quince collection containing 40 quince genotypes, originating from six distinct geographic areas, was screened using 15 SSR markers developed originally for apple and pear genomes. Overall, 13 markers exhibited polymorphism, with an average of 5.36 putative alleles per locus and a mean PIC value of 0.76. An UPGMA analysis divided the quince genotypes into five major clusters. The same results were obtained when the principal coordinates were plotted. The assignment test successfully allocated 83% of individuals into their place of origin. These results agree somewhat with the geographic origin of the quince accessions, and we conclude that geographic isolation leads to considerable genetic differentiation among Iranian quince collections. A significant ratio of transferability with a mean of 87.86% was measured, and we deduced that STMS markers derived from pear and apple have enough potential to detect polymorphism and differentiation in quince.

Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides.

An attempt was made to assess the expression level and targeting of a human protein entitled recombinant tissue plasminogen activator (rt-PA) through accumulation in three cellular compartments including the endoplasmic reticulum and cytosolic and apoplastic spaces in transgenic tobacco plants. In this context, three chimeric constructs pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA were employed and transferred into the tobacco plants through a popular transformation-based system called Agrobacterium tumefaciens. As an initial screening system, the incorporation of the rt-PA gene in the genomic DNA of tobacco transgenic plants and the possible existence of the rt-PA-specific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into the each transgenic plant; one or more copies were detected regarding transformants derived from all three abovementioned constructs. According to the enzyme-linked immunosorbent assay, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 µg/mg of the total soluble protein when a collection containing 30 transgenic plants transformed with pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA was taken into account, respectively. The zymography assay was lastly performed and concluded the expression of the properly folded rt-PA in this expression system. Our results, altogether, revealed that tobacco plants could be utilized as a bioreactor system for the large-scale production of enzymatically active t-PA and presumably other therapeutic recombinant proteins in large quantities.

Assessment of performance ability of three diagnostic methods for detection of Potato Leafroll virus (PLRV) using different visualizing systems.

To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC-RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC-RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC-RT-LAMP products, both hydroxynaphthol blue and GeneFinder™ could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. Altogether, as IC-RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.