Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme.

The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA.

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Manganese tolerance in Verbascum olympicum Boiss. affecting elemental uptake and distribution: changes in nicotinic acid levels under stress conditions.

A multielemental determination methodology in conjunction with an organic acid analysis that were supplemented with other stress parameters and an ultrastructural analysis used herein to study Verbascum olympicum Boiss. (Scrophulariaceae) under Mn stress. Uptake and accumulation characteristics of B, Cu, Fe, Mn, Mo, and Zn were evaluated in 8-week-old seedlings grown in Hoagland's nutrient solution and exposed to 5 (CK), 50, and 200 µM MnSO4 for 7 days. Hydrogen peroxide levels were determined to evaluate oxidative stress, and changes in compatible substance levels (total phenolic contents, glutathione and glutathione disulfide levels) were determined to assess antioxidant defense mechanisms. The distribution of manganese on the root surface was characterized by scanning electron microscopy images and energy-dispersive X-ray spectroscopy analysis. The levels of nicotinic acid, which is involved in nicotinamide adenine dinucleotide biosynthesis, were determined in roots and leaves to assess tolerance mechanisms. V. olympicum exhibited the ability to cope with oxidative stress originating from excessive Mn, while increased Mn concentrations were observed in both roots and leaves. The translocation factor of B was the most affected among other studied elements under the experimental conditions. Total nicotinic acid levels exhibited a trend of reduction in the roots and leaves, which could be attributed to the appropriate metabolic progress associated with oxidative stress based on the nicotinamide adenine dinucleotide cycle that may reach glutathione in response to manganese stress during plant growth.

Phytoremediation potential of wild plants growing on soil contaminated with heavy metals.

Phytoremediation is an emerging technology that employs higher plants to cleanup contaminated environments, including metal-polluted soils. Because it produces a biomass rich in extracted toxic metals, further treatment of this biomass is necessary. The aim of our study was to assess the five-year potential of the following native wild plants to produce biomass and remove heavy metals from a polluted site: poplar (Populus ssp.), ailanthus (Ailanthus glandulosa L.), false acacia (Robinia pseudoacacia L.), ragweed (Artemisia artemisiifolia L.), and mullein (Verbascum thapsus L). Average soil contamination with Pb, Cd, Zn, Cu, Ni, Cr, and As in the root zone was 22,948.6 mg kg-1, 865.4 mg kg-1, 85,301.7 mg kg-1, 3,193.3 mg kg-1, 50.7 mg kg-1, 41.7 mg kg-1,and 617.9 mg kg-1, respectively. We measured moisture and ash content, concentrations of Pb, Cd, Zn, Cu, Ni, Cr, and As in the above-ground parts of the plants and in ash produced by combustion of the plants, plus gross calorific values. The plants' phytoextraction and phytostabilisation potential was evaluated based on their bioconcentration factor (BCF) and translocation factor (TF). Mullein was identified as a hyperaccumulator for Cd. It also showed a higher gross calorific value (19,735 kJ kg-1) than ragweed (16,469 kJ kg-1).The results of this study suggest that mullein has a great potential for phytoextraction and for biomass generation, and that ragweed could be an effective tool of phytostabilisation.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

OBJECTIVES: To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. RESULTS: Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. CONCLUSION: Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Physiological basis of differential zinc and copper tolerance of Verbascum populations from metal-contaminated and uncontaminated areas.

Metal contamination represents a strong selective pressure favoring tolerant genotypes and leading to differentiation between plant populations. We investigated the adaptive capacity of early-colonizer species of Verbascum recently exposed to Zn- and Cu-contaminated soils (10-20 years). Two Verbascum thapsus L. populations from uncontaminated sites (NMET1, NMET2), one V. thapsus from a zinc-contaminated site (MET1), and a Verbascum lychnitis population from an open-cast copper mine (MET2) were exposed to elevated Zn or Cu in hydroponic culture under glasshouse conditions. MET populations showed considerably higher tolerance to both Zn and Cu than NMET populations as assessed by measurements of growth and net photosynthesis, yet they accumulated higher tissue Zn concentrations in the shoot. Abscisic acid (ABA) concentration increased with Zn and Cu treatment in the NMET populations, which was correlated to stomatal closure, decrease of net photosynthesis, and nutritional imbalance, indicative of interference with xylem loading and divalent-cation homeostasis. At the cellular level, the sensitivity of NMET2 to Zn and Cu was reflected in significant metal-induced ROS accumulation and ion leakage from roots as well as strong induction of peroxidase activity (POD, EC 1.11.1.7), while Zn had no significant effect on ABA concentration and POD activity in MET1. Interestingly, MET2 had constitutively higher root ABA concentration and POD activity. We propose that ABA distribution between shoots and roots could represent an adaptive mechanism for maintaining low ABA levels and unaffected stomatal conductance. The results show that metal tolerance can occur in Verbascum populations after relatively short time of exposure to metal-contaminated soil, indicating their potential use for phytostabilization.

Ni-induced Changes in Nitrate Assimilation and Antioxidant Metabolism of Verbascum olympicum Boiss.: Could the Plant be Useful for Phytoremediation or/and Restoration Purposes?

Verbascum olympicum Boiss. (Scrophulariaceae) were studied as a candidate plant for remediating the Ni polluted soils. The metabolic responses, such as nitrate assimilation (nitrate reductase and glutamine synthetase activity) and antioxidant system activity [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity], of this species exposed to nickel in Hoagland's nutrient medium were investigated as remediation performance parameters. The accumulation of nickel and the variations in the content of some elements (B, Cu, Fe, Mg, Mn, Mo and Zn) and some growth parameters, such as the water content, biomass production, and contents of chlorophyll and soluble protein, were also examined. The accumulation of Ni in both the roots and leaves varied depending on the exposure times and doses. Increased oxidative stress was suggested by the increases in the activities of SOD, CAT and APX. Although some element contents were inhibited by Ni treatments, these inhibitory effects was decreased depending on the time, and even these elements are accumulated in roots. These results are the novelties in the use of this species in biotechnology.

Chemical and mechanical defenses vary among maternal lines and leaf ages in Verbascum thapsus L. (Scrophulariaceae) and reduce palatability to a generalist insect.

Intra-specific variation in host-plant quality affects herbivore foraging decisions and, in turn, herbivore foraging decisions mediate plant fitness. In particular, variation in defenses against herbivores, both among and within plants, shapes herbivore behavior. If variation in defenses is genetically based, it can respond to natural selection by herbivores. We quantified intra-specific variation in iridoid glycosides, trichome length, and leaf strength in common mullein (Verbascum thapsus L, Scrophulariaceae) among maternal lines within a population and among leaves within plants, and related this variation to feeding preferences of a generalist herbivore, Trichopulsia ni Hübner. We found significant variation in all three defenses among maternal lines, with T. ni preferring plants with lower investment in chemical, but not mechanical, defense. Within plants, old leaves had lower levels of all defenses than young leaves, and were strongly preferred by T. ni. Caterpillars also preferred leaves with trichomes removed to leaves with trichomes intact. Differences among maternal lines indicate that phenotypic variation in defenses likely has a genetic basis. Furthermore, these results reveal that the feeding behaviors of T. ni map onto variation in plant defense in a predictable way. This work highlights the importance of variation in host-plant quality in driving interactions between plants and their herbivores.

Host dependent iridoid glycoside sequestration patterns in Cionus hortulanus.

Weevils of the genus Cionus (Curculionidae, Mecininae) sequester the iridoid glycosides (IGs) aucubin and catalpol from their host plants Scrophularia or Verbascum (Scrophulariaceae). Cionus hortulanus is the only member of the genus that feeds on both plant genera. We previously showed that sequestration patterns in C. hortulanus depend on the local host. To investigate whether IG patterns are driven by their availability in the hosts or genetic differences between populations, we collected C. hortulanus from S. nodosa in the field and reared them either on S. nodosa or on V. nigrum. The differences in IG concentrations were specific for the host plant upon which the weevils developed. Similar to monophagous species of the Cionini, individuals from S. nodosa had more aucubin than catalpol and mirrored the concentrations of their host plants. Specimens from V. nigrum, on the other hand, had higher concentrations of aucubin and of catalpol than their host. On V. nigrum, the ratio of catalpol to aucubin differed significantly between plant and beetle samples due to much higher catalpol concentrations in the weevils. Our data thus contradict genetically fixed differences between populations living on either plant but rather document the host plants' influence on the beetles' metabolism.

Combining optimal defense theory and the evolutionary dilemma model to refine predictions regarding plant invasion.

Optimal defense theory posits that plants with limited resources deploy chemical defenses based on the fitness value of different tissues and their probability of attack. However, what constitutes optimal defense depends on the identity of the herbivores involved in the interaction. Generalists, which are not tightly coevolved with their many host plants, are typically deterred by chemical defenses, while coevolved specialists are often attracted to these same chemicals. This imposes an "evolutionary dilemma" in which generalists and specialists exert opposing selection on plant investment in defense, thereby stabilizing defenses at intermediate levels. We used the natural shift in herbivore community composition that typifies many plant invasions to test a novel, combined prediction of optimal defense theory and the evolutionary dilemma model: that the within-plant distribution of defenses reflects both the value of different tissues (i.e., young vs. old leaves) and the relative importance of specialist and generalist herbivores in the community. Using populations of Verbascum thapsus exposed to ambient herbivory in its native range (where specialist and generalist chewing herbivores are prevalent) and its introduced range (where only generalist chewing herbivores are prevalent), we illustrate significant differences in the way iridoid glycosides are distributed among young and old leaves. Importantly, high-quality young leaves are 6.5x more highly defended than old leaves in the introduced range, but only 2x more highly defended in the native range. Additionally, defense levels are tracked by patterns of chewing damage, with damage restricted mostly to low-quality old leaves in the introduced range, but not the native range. Given that whole-plant investment in defense does not differ between ranges, introduced mullein may achieve increased fitness simply by optimizing its within-plant distribution of defense in the absence of certain specialist herbivores.

Metabolic differentiations and classification of Verbascum species by NMR-based metabolomics.

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of 1H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6-5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation.

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L(-1), respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.

Zinc-induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall.

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido-reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²+. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H2 DCFDA and EPR measurements. The extent of zinc-induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H2O2 production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, •CH3 and •OH, were found in the cell walls of zinc-treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell-wall-bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc-promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²+ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.

Verbascum bombyciferum Boiss. (Scrophulariaceae) as possible bio-indicator for the assessment of heavy metals in the environment of Bursa, Turkey.

In this study, we determined the heavy metal content (Cd(2+), Cr(3+), Cu(2+), Fe(3+), Ni(2+), Pb(2+), and Zn(2+)) in the soil surrounding the roots and different organs of Verbascum bombyciferum Boiss. (Scrophulariaceae), which is endemic to Uludag Mountain, Bursa, Turkey. Plant samples were collected from roadsides, and heavy metal accumulation capabilities were tested. This is one of the pioneer species of ruderal plant communities on roadsides, building sites, rubbish dumps, etc. Different organs of plant samples (roots, stems, leaves, and flowers) and their soils were analyzed by inductively couple plasma optical emission spectroscopy for their heavy metal contents. Some of the analyzed heavy metals (Cd(2+), Cr(3+), Pb(2+), and Zn(2+)) were usually increased depending on the traffic in the sample sites, and this variation was also reflected in heavy metal content of plant samples. Our results show that this plant can be used as a bio-indicator species in the monitoring of increased Cd(2+), Cr(3+), Pb(2+), and Zn(2+) in the environment. We also concluded that V. bombyciferum have the capability of Cd(2+), Cr(3+), Cu(2+), Ni(2+), Pb(2+), and Zn(2+) accumulation.

Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading.

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H(+) symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

Insecticidal properties of Verbascum cheiranthifolium against R. dominica on wheat and barley.

Tissues of higher plants contain novel natural substances that can be used to develop environmental safe methods for insect control. In this study, ethanol extract from flowers of Verbascum cheiranthifolium Boiss. (Scrophulariaceae) was examined for their effect on mortality and progeny production against adults of Rhyzopertha dominica (F.) on two commodities, wheat and barley. The botanical extract was applied at five dose rates, which 0.25, 0.5, 1.0, 2.0 and 3% (w/v). Adults of R. dominica were exposed to the treated wheat and peeled barley at 25 degrees C and 65% RH and mortality was assessed after 24 h, 48 h, 7 day, 14 day and 21 day of exposure. Then all adults were removed and the treated substrate remained at the same conditions for an additional 45 day after this interval, the commodities were checked for progeny production. In two commodities mortality increased with the increase of dose and exposure interval. Results indicated that on wheat, mortality was 100% after 14 days of exposure at the highest dose rate. Whereas, in the same conditions mortality of adults on barley was 63%. Thus plant extract was more effective against adults of R. dominica on wheat than application of barley. Interestingly in two diets, complete suppression (100%) of the progeny production was observed in the treated wheat and barley than in control even in the lowest dose rate.

Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides.

Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins.