Phylogenetic patterns of bioactive secondary metabolites produced by fungal endosymbionts in morning glories (Ipomoeeae, Convolvulaceae).

Heritable fungal endosymbiosis is underinvestigated in plant biology and documented in only three plant families (Convolvulaceae, Fabaceae, and Poaceae). An estimated 40% of morning glory species in the tribe Ipomoeeae (Convolvulaceae) have associations with one of two distinct heritable, endosymbiotic fungi (Periglandula and Chaetothyriales) that produce the bioactive metabolites ergot alkaloids, indole diterpene alkaloids, and swainsonine, which have been of interest for their toxic effects on animals and potential medical applications. Here, we report the occurrence of ergot alkaloids, indole diterpene alkaloids, and swainsonine in the Convolvulaceae; and the fungi that produce them based on synthesis of previous studies and new indole diterpene alkaloid data from 27 additional species in a phylogenetic, geographic, and life-history context. We find that individual morning glory species host no more than one metabolite-producing fungal endosymbiont (with one possible exception), possibly due to costs to the host and overlapping functions of the alkaloids. The symbiotic morning glory lineages occur in distinct phylogenetic clades, and host species have significantly larger seed size than nonsymbiotic species. The distinct and widely distributed endosymbiotic relationships in the morning glory family and their alkaloids provide an accessible study system for understanding heritable plant-fungal symbiosis evolution and their potential functions for host plants.

In vitro and in vivo antidiabetic activity, isolation of flavonoids, and in silico molecular docking of stem extract of Merremia tridentata (L.).

The antidiabetic activity of stem-ethanol extract (SE) and the flavonoid-rich fraction (FF) of Merremia tridentata (L.) were investigated on alloxan-induced diabetic mice. Apigenin, cosmosiin, and quercitrin are flavonoids isolated for the first time from stem extracts. In addition, cynaroside was found to be at the highest level in SE and FF with a percentage of 4.375% and 58.430%, respectively. The administration of SE (100 mg/kg) and FF (50, 75 mg/kg) daily for 20 days resulted in a better hypoglycemic effect than the reference drugs, glibenclamide (5 mg/kg), and metformin (10 mg/kg). Furthermore, SE and FF were shown to significantly improve the plasma lipid profiles at the end of the study. Docking's study suggests that cynaroside, cosmosiin, and quercitrin are the most desirable compounds for hypoglycemic effects in many antidiabetic targets. Especially, SE and FF showed strongly α-amylase and α-glucosidase inhibitory activities (IC50 = 1.61-1.72 mg/mL on α-amylase and IC50 = 0.24-0.44 mg/mL on α-glucosidase). Therefore, SE and FF of Merremia tridentata is a potential drug with antidiabetic and hypoglycemic action as indicated by in vivo, in silico, and in vitro studies.

Assessment of phytoremediation capacity of three halophytes: Suaeda monoica, Tamarix indica and Cressa critica.

Halophyte is a distinctive group of plants that can survive, even well flourish, at a concentration of Na+ and Cl- ions along with heavy metals that would be lethal to most of the agricultural crop species. These capabilities make certain halophytes good contenders for phytoremediation through phytoextraction or phytostabilization of the salt and heavy metals (HMs) in polluted soils. Thus, the present study elucidates the phytoextraction capacity of three halophytes (Suaeda monoica, Tamarix indica and Cressa critica) growing in saline soil (EC 112 ds m-1), with higher level of HMs rather than a cultivated soil. The accumulation of ions in above-ground tissue was determined in the all three studied plants, considering the fact that maintaining a stable cytosolic Na+/K+ ratio has become a crucial salinity tolerance mechanism. The higher salinity of soil resulted in high level of Na+ ions in leaves, increased synthesis of osmolyte components and robust antioxidant activities to combat the oxidative stress. As whole, changes in cellular metabolites were determined by using FT-IR spectroscopy, evident as differential FT-IR profiles in both leaves and stem specific to these metabolites. The considerable amounts of HMs accumulation including Zn, Fe, Mn, Cu, Cr, and Cd with highest being Fe in above-ground tissue of all three studied halophytes were obtained. These preliminary findings represent S. monoica, T. indica and C. cretica as potent phytoremediation plant using phytosequestration to accumulate HMs. The present study project a light on the use of these three plants in reclamation of degraded saline soils.

Depth related adaptations in symbiont bearing benthic foraminifera: New insights from a field experiment on Operculina ammonoides.

Large benthic foraminifera (LBF) are marine calcifying protists that commonly harbor algae as symbionts. These organisms are major calcium carbonate producers and important contributors to primary production in the photic zones. Light is one of the main known factors limiting their distribution, and species of this group developed specific mechanisms that allow them to occupy different habitats across the light gradient. Operculina ammonoides (Gronovius, 1781) is a planispiral LBF that has two main shell morphotypes, thick involute and flat evolute. Earlier studies suggested morphologic changes with variation in water depth and presumably light. In this study, specimens of the two morphotypes were placed in the laboratory under artificial low light and near the sea floor at depths of 15 m, 30 m, and 45 m in the Gulf of Aqaba-Eilat for 23 days. Differences in growth and symbionts content were evaluated using weight, size, and chlorophyll a. Our results show that O. ammonoides exhibit morphological plasticity when constructing thinner chambers after relocation to low light conditions, and adding more weight per area after relocation to high light conditions. In addition, O. ammonoides exhibited chlorophyll content adaptation to a certain range of light conditions, and evolute specimens that were acclimatized to very low light did not survive relocation to a high light environment, possibly due to photo-oxidative stress.

Compounds Identification in Semen Cuscutae by Ultra-High-Performance Liquid Chromatography (UPLCs) Coupled to Electrospray Ionization Mass Spectrometry.

Semen Cuscutae is commonly used in traditional Chinese medicine and contains a series of compounds such as flavonoids, chlorogenic acids and lignans. In this study, we identified different kinds of compositions by ultra-high-performance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry (MS). A total of 45 compounds were observed, including 20 chlorogenic acids, 23 flavonoids and 2 lignans. 23 of them are reported for the first time including 6-O-caffeoyl-ß-glucose, 3-O-(4'-O-Caffeoylglucosyl) quinic acid, etc. Their structures were established by retention behavior, extensive analyses of their MS spectra and further determined by comparison of their MS data with those reported in the literature. As chlorogenic acids and flavonoids are phenolic compounds that are predominant in Semen Cuscutae, in conclusion, phenolic compounds are the major constituents of Semen Cuscutae.

A facile and green strategy for the synthesis of Au, Ag and Au-Ag alloy nanoparticles using aerial parts of R. hypocrateriformis extract and their biological evaluation.

A facile and green strategy is reported here to synthesize gold (Au), silver (Ag) and gold-silver (Au-Ag) alloy nanoparticles (NPs) through bio-reduction reactions of aqueous corresponding metal precursors mediated by extracts of aerial parts of R. hypocrateriformis, which act as both reducing and stabilizing agents, under microwave irradiation. UV-vis spectrophotometer, XRD, FT-IR, FESEM/TEM, TGA and EDAX analysis were used to characterize the obtained NPs. The formation of NPs is evident from their surface plasmon resonance peak observed at λmax=∼550, 450 and 500nm for Au, Ag and Au-Ag alloy NPs respectively. XRD pattern revealed that fcc structure, while FT-IR spectra signify the presence of phytochemicals adsorbed on NPs. Such a biofunctionalized NPs were characterized by their weight loss, 30% due to thermal degradation of plant phytochemicals observed in TG analysis. The spherical shape of Au, Ag and Au-Ag alloy NPs (∼10-50nm) is observed by FE-SEM/TEM images. EDAX analysis confirms the expected elemental composition. Moreover, these NPs showed enhanced antimicrobial, antioxidant, and anticancer activities, though it is more pronounced for Au-Ag alloy NPs, which is due to the combining effect of phytochemicals, Au and Ag metals. Thus, the biosynthesized NPs could be applied as effective growth inhibitors for various biomedical applications.

In vitro propagation and cell cultures of memory tonic herb Evolvulus alsinoides: a best source for elicited production of scopoletin.

Evolvulus alsinoides L. is used for preparation of 'Shankhapushpi', an important popular ayurvedic drug that contributes considerably to the improvement of memory power. The improvement is attributed to the presence of furanocoumarin scopoletin, a metabolite with a wide range of biological activities. This report describes, for the first time, an in vitro culture system for propagation and enhanced production of scopoletin. Different concentrations of auxins and cytokinins individually and in combination were used in Murashige and Skoog (MS) medium to induce shoot regeneration in cotyledonary nodal explants and callus formation in leaf explants. The best response was achieved in MS medium fortified with 5.0 µM 6-benzyladenine (BA) in which 96 % of cultures produced 7.6 ± 0.6 shoots per explant. Regenerated shoots were rooted on MS medium with 5.0 µM indole-3-acetic acid (IAA). Plantlets were successfully acclimatized and established in soil. MS medium fortified with 10 µM BA + 5.0 µM IAA showed maximum growth and accumulation of scopoletin in cell cultures. Cell cultures could be maintained over 24 months. The influences of auxins, cytokinins, organic acids, amino acids, and fungal-derived elicitors on production of scopoletin were studied. Presence of either L-arginine, sodium pyruvate, or yeast extract highly promoted scopoletin production as compared with control and achieved 75.02-, 72.13-, and 57.98-fold higher accumulation, respectively. The results presented herein have laid solid foundation for large-scale production of scopoletin and further investigation of its purification and utilization as a novel pharmaceutical drug.

Flavonoids and their qualitative variation in Calystegia soldanella and related species (Convolvulaceae).

Coastal species are exposed to severe environmental stresses, e.g. salt and UV-B. The plants adapt themselves to such harsh environment by controlling morphological features and chemical defense systems. Flavonoids are known as efficient anti-stress polyphenols produced by plants. Most flavonoids show antioxidant activity, and their properties are important for plants to survive under high-stress conditions such as those in a coastal area. Among the compounds, ortho-dihydroxylated flavonoids act as strong antioxidants. In this survey, we elucidated the flavonoid composition of a seashore species Calystegia soldanella, which is distributed not only on the seashore, but also by the inland freshwater lake, Lake Biwa. Seven flavonol glycosides, i.e. quercetin 3-0- rutinoside, 3-O-glucoside, 3-O-rhamnoside and 3-O-apiosyl-(1-->2)-[rhamnosyl-(1-->6)-glucoside], and kaempferol 3-O-rutinoside, 3-O-glucoside and 3-0- rhamnoside were isolated from the leaves of C. soldanella. In addition, it was shown that the quercetin (Qu) to kaempferol (Km) ratio of coastal populations was higher than that of lakeshore populations. In general, these differences of Qu/Km ratio depend on flavonoid 3'-hydroxylase (F3'H) transcription. RT-PCR analysis suggested that F3'H of C. soldanella is regulated translationally or post-translationally, but not transcriptionally. Furthermore, quantitative and qualitative differences in flavonoid composition occurred among three Calystegia species, C. soldanella, C. japonica and C. hederacea.

Chemical composition and aroma evaluation of essential oils from Evolvulus alsinoides L.

The aim of this study was to investigate the chemical composition and the odor-active compounds of the essential oils from Evolvulus alsinoides, which is a well-known edible and medicinal plant. The volatile compounds in the oils were identified by hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE) in combination with GC, GC/MS, GC/O (=olfactometry), aroma extract dilution analysis (AEDA), and relative flavor activities (RFA values). The most abundant compound in the HD oil was cis-α-necrodol (12.62%), an irregular monoterpene with a cyclopentane skeleton, which is very unusual in the plant kingdom. In the SAFE oil, the main components included 2-butoxyethanol (9.01%), benzyl alcohol (8.01%), and γ-butyrolactone (7.37%). Through sensory analysis, 21 aroma-active compounds were identified by GC/O. The most intense aroma-active compounds in the HD oil were hexan-1-ol and γ-nonalactone, both of which showed high RFA values. α-Methyl-γ-butyrolactone and dimethyl sulfone contributed more strongly to the aroma of the SAFE oil. These results imply that the essential oils of E. alsinoides deserve further investigation in the food industry.

Physical dormancy in seeds of the holoparasitic angiosperm Cuscuta australis (Convolvulaceae, Cuscuteae): dormancy-breaking requirements, anatomy of the water gap and sensitivity cycling.

BACKGROUND AND AIMS: Dormancy in seeds of Cuscuta (Convolvulaceae, tribe Cuscuteae) is due to a water-impermeable seed coat (physical dormancy). In nondormant seeds of several species of this family, bulges adjacent to the micropyle have been identified as the initial route of water entry into seeds (water gap). However, there are claims that water enters seeds of Cuscuta spp. via the entire seed coat. Although several studies have been done on seed coat anatomy of Cuscuta, none has identified and/or characterized the morphology/anatomy of a water gap. Thus, the primary aim of this research was to identify and describe the morphology and anatomy of the water gap in seeds of Cuscuta australis. It was also determined if sensitivity cycling to dormancy-breaking treatments occurs in seeds of this species. METHODS: Light microscopy, scanning electron microscopy, tissue-sectioning and dye-tracking and blocking experiments were used to investigate the morphology and anatomy of the water gap. Treatments simulating natural conditions were used to break seed dormancy. Storage of seeds at different temperatures was tested for their effect on sensitivity to dormancy-breaking treatment. KEY RESULTS: Dormancy-breaking treatments caused the tightly closed hilar fissure to open. Staining was observed in cells below the hilum area but not in those below the seed coat away from the hilum. Sensitivity to dormancy-breaking treatment was induced by storing seeds dry and reduced by storing them wet. CONCLUSIONS: Whereas bulges adjacent to the micropyle act as the water gap in other species of Convolvulaceae with physical dormancy, the hilar fissure serves this function in Cuscuta. Cuscuta australis can cycle between insensitivity <--> sensitivity to dormancy-breaking treatments.

Clavicipitaceous fungi associated with ergoline alkaloid-containing convolvulaceae.

Ergoline alkaloids are a group of physiologically active natural products occurring in taxonomically unrelated fungal and plant taxa Clavicipitaceae (Hypocreales) and Convolvulaceae (Solanales). We show in the present paper that clavicipitaceous fungi are associated with four different ergoline alkaloid-containing plant taxa of the family Convolvulaceae. These fungi are macroscopically visible on the adaxial surface when young leaf buds are opened or are detectable by molecular biological techniques in seeds. Detectability of the fungus correlates with the absence or presence of ergoline alkaloids within the respective plant organ. The fungi contain the gene (dmaW) responsible for the committed step in ergoline alkaloid biosynthesis. Sequencing of ribosomal DNA (18S rDNA and internal transcribed spacer) as well as the dmaW gene (partial) and construction of phylogenetic trees show that the fungi are clavicipitaceous, not identical but very closely related.

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil.

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil.

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.

Characterization of two tomato aquaporins and expression during the incompatible interaction of tomato with the plant parasite Cuscuta reflexa.

A subtractive suppression hybridization technique was used to identify genes that were induced during early phases of the interaction between Cuscuta reflexa, a phanerogamic plant parasite and the incompatible host tomato (Lycopersicon esculentum Mill.). One of the identified genes encodes a new aquaporin (LeAqp2) from tomato. Its function was concluded from the swelling kinetics of LeAqp2-expressing Xenopus laevis oocytes under hypo-osmotic conditions. It was shown that, 6 h after attachment of the plant parasite, the corresponding mRNA accumulated in cells at and adjacent to the attachment site of Cuscuta, while artificial wounding did not modify steady-state LeAqp2- RNA levels. Expression of a close homologue named TRAMP (tomato-ripening-associated protein) was not affected by the plant-plant interaction. Levels of indole-3-acetic acid (IAA) in tomato tissue after infection by Cuscuta have been found to increase at a similar stage of infection. In contrast to the different behavior with respect to infection, IAA induced both LeAqp2 and TRAMP expression. The observed pattern of LeAqp2 expression during the interaction at a stage where cell elongation occurs together with the water-channel activity in the heterologous expression system suggest a function for LeAqp2 during the tomato-Cuscuta interaction.