Carnivorous Plants from Nepenthaceae and Droseraceae as a Source of Secondary Metabolites.

Carnivorous plants are able to attract small animals or protozoa and retain them in their specialized traps. Later, the captured organisms are killed and digested. The nutrients contained in the prey bodies are absorbed by the plants to use for growth and reproduction. These plants produce many secondary metabolites involved in the carnivorous syndrome. The main purpose of this review was to provide an overview of the secondary metabolites in the family Nepenthaceae and Droseraceae, which were studied using modern identification techniques, i.e., high-performance liquid chromatography or ultra-high-performance liquid chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. After literature screening, there is no doubt that tissues of species from the genera Nepenthes, Drosera, and Dionaea are rich sources of secondary metabolites that can be used in pharmacy and for medical purposes. The main types of the identified compounds include phenolic acids and their derivatives (gallic, protocatechuic, chlorogenic, ferulic, p-coumaric acids, gallic, hydroxybenzoic, vanillic, syringic caffeic acids, and vanillin), flavonoids (myricetin, quercetin, and kaempferol derivatives), including anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, and cyanidin), naphthoquinones (e.g., plumbagin, droserone, and 5-O-methyl droserone), and volatile organic compounds. Due to the biological activity of most of these substances, the importance of the carnivorous plant as a pharmaceutical crop will increase.

Asian Ancistrocladus Lianas as Creative Producers of Naphthylisoquinoline Alkaloids.

This book describes a unique class of secondary metabolites, the mono- and dimeric naphthylisoquinoline alkaloids. They occur in lianas of the paleotropical Ancistrocladaceae and Dioncophyllaceae families, exclusively. Their unprecedented structures include stereogenic centers and rotationally hindered, and thus likewise stereogenic, axes. Extended recent investigations on six Ancistrocladus species from Asia, as reported in this review, shed light on their fascinating phytochemical productivity, with over 100 such intriguing natural products. This high chemodiversity arises from a likewise unique biosynthesis from acetate-malonate units, following a novel polyketidic pathway to plant-derived isoquinoline alkaloids. Some of the compounds show most promising antiparasitic activities. Likewise presented are strategies for the regio- and stereoselective total synthesis of the alkaloids, including the directed construction of the chiral axis.

Structural Studies on Diverse Betacyanin Classes in Matured Pigment-Rich Fruits of Basella alba L. and Basella alba L. var. 'Rubra' (Malabar Spinach).

Identification of betacyanins in Basella alba L. and Basella alba L. var. 'Rubra' fruits was performed by low- and high-resolution mass spectrometry (LRMS and HRMS) as well as 1H, 13C and two-dimensional NMR which revealed hitherto completely not known betacyanin classes in the plant kingdom. Especially, the presence of unique nitrogenous acyl moieties in the structures of the pigments was ascertained by the HRMS Orbitrap detection. Except for detected polar betacyanin glycosylated derivatives, presence of a series of previously not reported pigments such as malonylated betanidin 6-O-ß-glusosides with their acyl migration isomers along with the evidence of the 3''-hydroxy-butyrylated betacyanins is reported. The first complete NMR data were obtained for novel and principal acylated gomphrenins with hydroxycinnamic acids: 6'-O-E-caffeoyl-gomphrenin (malabarin), 6'-O-E-sinapoyl-gomphrenin (gandolin), 6'-O-E-4-coumaroyl-gomphrenin (globosin) and 6'-O-E-feruloyl-gomphrenin (basellin).

Evaluation of the Effect of Elite Jojoba Lines on the Chemical Properties of their Seed Oil.

Jojoba oil (JO) extracted from seeds has outstanding properties, including anti-inflammatory, antioxidant, and antibacterial activities, and can be stored forlong periodsof time. The unique properties of jojoba oil depend on its chemical composition; therefore, the effect of the jojoba genotype on the chemical properties and active components of the seed oil was evaluated in this study. Oil samples were collected from 15 elite Egyptian jojoba lines. The chemical composition, such as moisture, crude fiber, crude oil, ash, and crude protein of elite lines' seeds was determined to investigate the variation among them based on the jojoba genotype. In addition, the iodine value was obtained to measure the degree of jojoba oil unsaturation, whereas the peroxide number was determined as an indicator of the damage level in jojoba oil. Fatty acid composition was studied to compare elite jojoba lines. Fatty acid profiles varied significantly depending on the jojoba genotype. Gadoleic acid exhibited the highest percentage value (67.85-75.50%) in the extracted jojoba oil, followed by erucic acid (12.60-14.81%) and oleic acid (7.86-10.99%). The iodine value, peroxide number, and fatty acid composition of the tested elite jojoba lines were compared withthose reported by the International Jojoba Export Council (IJEC). The results showed that the chemical properties of jojoba oils varied significantly, depending on the jojoba genotype.

Phytochemical Profiling, Antioxidant, Anti-Inflammatory, Thrombolytic, Hemolytic Activity In Vitro and In Silico Potential of Portulacaria afra.

The use of complementary herbal medicines has recently increased in an attempt to find effective alternative therapies that reduce the adverse effects of chemical drugs. Portulacaria afra is a rich source of phytochemicals with high antioxidant activity, and thus may possess health benefits. This study used the latest developments in GC-MS coupling with molecular docking techniques to identify and quantify the phytoconstituents in P. afra tissue extracts. The results revealed that n-butanol P. afra (BUT-PA) dry extracts contained total phenolic and flavonoids contents of 21.69 ± 0.28 mgGAE/g and 196.58 ± 6.29 mgGAE/g, respectively. The significant potential of antioxidants was observed through CUPRIC, FRAP, and ABTS methods while the DPPH method showed a moderate antioxidants potential for P. afra. Enzymatic antioxidants, superoxide dismutase, peroxidase and catalase also showed a better response in the BUT-PA dry extracts. The thrombolytic activity of the BUT-PA extracts ranged from 0.4 ± 0.32 to 11.2 ± 0.05%. Similarly, hemolytic activity ranged from 5.76 ± 0.15 to 9.26 ± 0.15% using the standard (triton x) method. The BUTPA and CHPA showed moderate acetylcholinesterase and butrylcholinesterase inhibition, ranging from 40.78 ± 0.52 to 58.97 ± 0.33, compared to galantamine. The carrageenan induced hind-paw edema assay, while BUT-PA extracts showed anti-inflammatory properties in a dose-dependent manner. Furthermore, 20 compounds were identified in the BUTPA extracts by GC-MS. Molecular docking was performed to explore the synergistic effect of the GC-MS-identified compounds on COX-1 and COX-2 inhibition. A high binding affinity was observed for Stigmastan-3, 5-diene, Phthalic acid, 3. Alpha-Hydroxy-5, 16-androstenol. The computed binding energies of the compounds revealed that all the compounds have a synergistic effect, preventing inflammation. It was concluded that active phytochemicals were present in P. afra, with the potential for multiple pharmacological applications as a latent source of pharmaceutically important compounds. This should be further explored to isolate secondary metabolites that can be employed in the treatment of different diseases.

Structural characterization of the carbohydrate and protein part of arabinogalactan protein from Basella alba stem and antiadhesive activity of polysaccharides from B. alba against Helicobacter pylori.

BACKGROUND: Increasing drug resistance of Helicobacter pylori has highlighted the search for natural compounds with antiadhesive properties, interrupting the adhesion of H. pylori to stomach epithelia. Basella alba, a plant widely used in Asian traditional medicine, was investigated for its antiadhesive activity against H. pylori. METHODS: B. alba extract FE was prepared by aqueous extraction. Polysaccharides were isolated from FE by ethanol precipitation and arabinogalactan-protein (AGP) was isolated with Yariv reagent. Carbohydrate analyses was performed by standard methods and sequence analysis of the protein part of AGP by LC-MS. In vitro adhesion assay of fluorescent-labelled H. pylori J99 to human AGS cells was performed by flow cytometric analysis. RESULTS: Raw polysaccharides (BA1) were isolated and 9% of BA1 were identified as AGP (53.1% neutral carbohydrates L-arabinose, D-galactose, rhamnose, 5.4% galacturonic acid, 41.5% protein). After deglycosylation of AGP, the protein part (two bands at 15 and 25 kDa in tricine SDS-PAGE) was shown to contain peptides like ribulose-bisphosphate-carboxylase-large-chain. Histological localization within the stem tissue of B. alba revealed that AGP was mainly located at the procambium ring. Functional assays indicated that neither FE nor BA1 had significant influence on viability of AGS cells or on H. pylori. FE inhibited the bacterial adhesion of H. pylori to AGS cells in a dose dependent manner. Best anti-adhesive effect of ~67% was observed with BA1 at 2 mg/mL. CONCLUSION: The data obtained from this study characterize in part the mucilage and isolated polysaccharides of B. alba. As the polysaccharides interact with the bacterial adhesion, a potential uses a supplemental antiadhesive entity against the recurrence of H. pylori after eradication therapy may be discussed.

Sodium hyperaccumulators in the Caryophyllales are characterized by both abnormally large shoot sodium concentrations and [Na]shoot/[Na]root quotients greater than unity.

BACKGROUND AND AIMS: Some Caryophyllales species accumulate abnormally large shoot sodium (Na) concentrations in non-saline environments. It is not known whether this is a consequence of altered Na partitioning between roots and shoots. This paper tests the hypotheses (1) that Na concentrations in shoots ([Na]shoot) and in roots ([Na]root) are positively correlated among Caryophyllales, and (2) that shoot Na hyperaccumulation is correlated with [Na]shoot/[Na]root quotients. METHODS: Fifty two genotypes, representing 45 Caryophyllales species and 4 species from other angiosperm orders, were grown hydroponically in a non-saline, complete nutrient solution. Concentrations of Na in shoots and in roots were determined using inductively coupled plasma mass spectrometry (ICP-MS). KEY RESULTS: Sodium concentrations in shoots and roots were not correlated among Caryophyllales species with normal [Na]shoot, but were positively correlated among Caryophyllales species with abnormally large [Na]shoot. In addition, Caryophyllales species with abnormally large [Na]shoot had greater [Na]shoot/[Na]root than Caryophyllales species with normal [Na]shoot. CONCLUSIONS: Sodium hyperaccumulators in the Caryophyllales are characterized by abnormally large [Na]shoot, a positive correlation between [Na]shoot and [Na]root, and [Na]shoot/[Na]root quotients greater than unity.

Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.

Natural or light-induced pigment accumulation in grain amaranths coincides with enhanced resistance against insect herbivory.

MAIN CONCLUSION: Increased resistance to insect herbivory in grain amaranth plants is associated with increased betalain pigmentation, either naturally acquired or accumulated in response to blue-red light irradiation. Betalains are water-soluble pigments characteristic of plants of the Caryophyllales order. Their abiotic stress-induced accumulation is believed to protect against oxidative damage, while their defensive function against biotic aggressors is scarce. A previous observation of induced betalain-biosynthetic gene expression in stressed grain amaranth plants led to the proposal that these pigments play a defensive role against insect herbivory. This study provided further support for this premise. First, a comparison of "green" and "red" Amaranthus cruentus phenotypes showed that the latter suffered less insect herbivory damage. Coincidentally, growth and vitality of Manduca sexta larvae were more severely affected when fed on red-leafed A. cruentus plants or on an artificial diet supplemented with red-leaf pigment extracts. Second, the exposure of A. cruentus and A. caudatus plants, having contrasting pigmentation phenotypes, to light enriched in the blue and red wavelength spectra led to pigment accumulation throughout the plant and to increased resistance to insect herbivory. These events were accompanied by the induced expression of known betalain-biosynthetic genes, including uncharacterized DODA genes believed to participate in this biosynthetic pathway in a still undefined way. Finally, transient co-expression of different combinations of betalain-biosynthetic genes in Nicotiana benthamiana led to detectable accumulation of betalamic acid and betanidin. This outcome supported the participation of certain AhDODA and other genes in the grain amaranth betalain-biosynthetic pathway.

Talinum paniculatum (Jacq.) Gaertn. leaves - source of nutrients, antioxidant and antibacterial potentials.

BACKGROUND: The diet of most of the population is limited to a reduced number of plants, even in areas that have a varied and extensive diversity, such as Brazil. Unconventional Food Plants (UFPs) are plants considered exotic, native, and wild that grow naturally and can be used as food. Among these is Talinum paniculatum (Jacq.) Gaertn., which is widespread throughout Brazil and can be a potential source of nutrients. Due to the potential of utilization of UFPs in human food and the lack of studies regarding the composition of T. paniculatum, this study aimed to assess the nutritional value of T. paniculatum leaves, their antioxidant capacity, and their antimicrobial activity for possible use in food. METHODS: The characterization of the leaves of T. paniculatum was carried out through analyses of proximal composition, color, ascorbic acid, mineral profile, and antinutritional factors showing the presence of condensed and hydrolysable tannins and nitrates in low concentrations. Solvents of water, ethanol, ethanol/water, methanol, methanol/water, methanol/acetic acid and acetone/water/acetic acid were used to evaluate the extraction yield of phenolic compounds, antioxidant capacity, and antibacterial activity of the extracts. RESULTS: High contents of protein (18.61 g 100 g-1), insoluble dietary fiber (34.75 g 100 g-1), ascorbic acid (81.03 mg 100 g-1), magnesium, potassium, and calcium (649.600, 411.520 and 228.117 mg 100 g-1, respectively) were observed. Extraction using the mixture of solvents of methanol/acetic acid showed the highest yield of phenolic compounds (432.73 mg EAG 100 g-1) and antioxidant capacity using the DPPH assay (3144.92 mg 100 g-1). Bacillus cereus growth was inhibited by the T. paniculatum extracts. CONCLUSIONS: T. paniculatum leaves are a source of nutrients and their extracts have antioxidant and antibacterial potentials which can be used as supplements in food to improve one's health.

N,C-Coupled naphthylisoquinoline alkaloids: a versatile new class of axially chiral natural products.

Covering: up to April 2021During the past decades, a plethora of natural products with restricted rotation about a biaryl axis have been discovered, among them the naphthylisoquinoline (NIQ) alkaloids, mostly C,C-coupled and having remarkable bioactivities. Within this fascinating class of naturally occurring biaryl compounds, NIQ alkaloids bearing an N,C-heterobiaryl axis have attracted particular attention. They are structurally and biosynthetically unprecedented, with interesting stereochemical implications and biological activities. In contrast to existing articles and reviews about axially chiral - yet C,C-coupled - natural products, this is the first, comprehensive review on the new subclass of N,C-coupled NIQs, their isolation and structural elucidation, their N,C-axial chirality, their biosynthetic origin, their promising antiparasitic and antileukemic activities, and their total synthesis.

Optimization of microwave assisted extraction of simmondsins and polyphenols from Jojoba (Simmondsia chinensis) seed cake using Box-Behnken statistical design.

A closed-vessel microwave-assisted extraction (MAE) of simmondsins and polyphenols from defatted Jojoba cake using Box-Benkhen design with four independent variables (solvent/cake ratio, ethanol concentration, extraction time and microwave power) was investigated. ANOVA results showed that the obtained models were significant at 95% confidence level. Optimal extraction conditions were found for highest values of microwave power (500 W) and extraction time (15 min) and for moderate values of solvent to cake ratio (41 - 45 mL/g). Optimum simmondsins yield (23.35%) was obtained with pure water as solvent. However, optimum polyphenols yield (2.33%) and ORAC antioxidant activity (656 µmol TE/g) were obtained with 46.79% and 42.04% ethanol in water, respectively. ORAC antioxidant activity was found to be well correlated to polyphenol and simmondsin contents. These results indicate that MAE is an effective technique for recovery of bioactive compounds for food and pharmaceutical industries from Jojoba by-products.

CAM photosynthesis in desert blooming Cistanthe of the Atacama, Chile.

When plants of the Atacama desert undergo episodic blooms, among the most prominent are succulent-leaved Cistanthe (Montiaceae). We demonstrate that two Cistanthe species, the perennial Cistanthe sp. aff. crassifolia and the annual/biannual Cistanthe sp. aff. longiscapa, can exhibit net CO2 uptake and leaf acidification patterns typical of crassulacean acid metabolism (CAM). In C. sp. aff. crassifolia leaves, CAM expression was facultative. CAM-type nocturnal net CO2 uptake and acid accumulation occurred in drought-stressed but not in well-watered plants. By contrast, CAM expression in C. sp. aff. longiscapa was largely constitutive. Nocturnal acid accumulation was present in leaves of well-watered and in droughted plants. Following water-deficit stress, net nocturnal CO2 uptake was induced and the level of acid accumulated increased. Neither nocturnal CO2 uptake nor acid accumulation was reduced when the plants were re-watered. δ13C values of a further nine field-collected Cistanthe species are consistent with a contribution of CAM to their carbon pools. In the Portulacinae, a suborder with eight CAM-containing families, Cistanthe becomes the sixth genus with CAM within the family Montiaceae, and it is likely that the ancestor of all Portulacineae also possessed CAM photosynthesis. In the stochastic rainfall landscape of the Atacama, carbon uptake in the dark is a water-use efficient mechanism that increases the carbon pool available for seed production or dormancy. The next rain event may be years away.

Spirombandakamine A3 and Cyclombandakamines A8 and A9, Polycyclic Naphthylisoquinoline Dimers, with Antiprotozoal Activity, from a Congolese Ancistrocladus Plant.

Spirombandakamine A3 (7) is only the third known naphthylisoquinoline dimer with a spiro-fused novel molecular framework and the first such representative to possess a relative trans-configuration at the two chiral centers in both tetrahydroisoquinoline subunits. It was found in the leaves of a botanically as yet unidentified Congolese Ancistrocladus plant, which is morphologically closely related to the Central African taxon Ancistrocladus ealaensis. Likewise isolated were the new cyclombandakamines A8 (8) and A9 (9), which belong to another most recently discovered type of unusual oxygen-bridged naphthylisoquinoline dimers and two previously described "open-chain" analogues, mbandakamines C (10) and D (11). The full absolute stereostructures of these compounds were assigned by combining spectroscopic, chemical, and chiroptical methods. Preliminary biomimetic investigations indicated that both spirombandakamine- and cyclombandakamine-type dimers result from the oxidation of their open-chain mbandakamine-type congeners. The new dimeric alkaloids 7-9 displayed potent growth-inhibitory activity against Plasmodium falciparum, the protozoal pathogen causing malaria, and moderate effects on Trypanosoma brucei rhodesiense, the parasite responsible for African sleeping sickness.

A biome-wide experiment to assess the effects of propagule size and treatment on the survival of Portulacaria afra (spekboom) truncheons planted to restore degraded subtropical thicket of South Africa.

Insights from biome-wide experiments can improve efficacy of landscape-scale ecological restoration projects. Such insights enable implementers to set temporal and geographical benchmarks and to identify key drivers of success during the often decades-long restoration trajectory. Here we report on a biome-wide experiment aimed at informing the ecological restoration of thousands of hectares of degraded subtropical thicket dominated by the succulent shrub, Portulacaria afra (spekboom). Restoration using spekboom truncheons has the potential to sequester, for a semi-arid region, large amounts of ecosystem carbon, while regenerating a host of associated ecosystem services. This study evaluates, after about three years post-propagation, the effects of spekboom truncheon size and treatment on survivorship in 40 fence-enclosed (0.25 ha) plots located in target habitat across the entire spekboom thicket biome. In each plot, locally harvested spekboom truncheons, comprising eight size/treatment combinations, were planted in replicated rows of between 24 and 49 individuals, depending on treatment. The experiment assessed the role of truncheon size, spacing, application of rooting hormone and watering at planting on survivorship percentage as an indicator of restoration success. All eight combinations recorded extreme minimum survivorship values of zero, while the range of extreme maximum values was 70-100%. Larger truncheons (>22.5 mm diameter) had almost double the survivorship (ca. 45%) than smaller truncheons (< 15 mm) (ca. 25%). Planting large, untreated truncheons at 1 m intervals-as opposed to 2 m intervals recommended in the current restoration protocol-resulted in no significant change in survivorship. The application of rooting hormone and water at planting had no significant effect on restoration success for both large and small truncheons. While our results do not provide an evidence base for changing the current spekboom planting protocol, we recommend research on the financial and economic costs and benefits of different propagation strategies in real-world contexts.

Plumbagin, a Potent Naphthoquinone from Nepenthes Plants with Growth Inhibiting and Larvicidal Activities.

Some plant species are less susceptible to herbivore infestation than others. The reason for this is often unknown in detail but is very likely due to an efficient composition of secondary plant metabolites. Strikingly, carnivorous plants of the genus Nepenthes show extremely less herbivory both in the field and in green house. In order to identify the basis for the efficient defense against herbivorous insects in Nepenthes, we performed bioassays using larvae of the generalist lepidopteran herbivore, Spodoptera littoralis. Larvae fed with different tissues from Nepenthes x ventrata grew significantly less when feeding on a diet containing leaf tissue compared with pitcher-trap tissue. As dominating metabolite in Nepenthes tissues, we identified a naphthoquinone, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone). When plumbagin was added at different concentrations to the diet of S. littoralis larvae, an EC50 value for larval growth inhibition was determined with 226.5 µg g-1 diet. To further determine the concentration causing higher larval mortality, sweet potato leaf discs were covered with increasing plumbagin concentrations in no-choice-assays; a higher mortality of the larvae was found beyond 60 µg plumbagin per leaf, corresponding to 750 µg g-1. Plant-derived insecticides have long been proposed as alternatives for pest management; plumbagin and derivatives might be such promising environmentally friendly candidates.

The Development of a Phytopathogenic Fungi Control Trial: Aspergillus flavus and Aspergillus niger Infection in Jojoba Tissue Culture as a Model.

After introducing the idea of using concentrations equal to or less than the minimum inhibition concentration (MIC) of some active chemical compounds for evacuating microbial cells, different types of microbes were evacuated. The original protocol was given the name sponge-like protocol and then was reduced and modified from a microorganism to another to prepare microbial ghosts for various applications such as immunological applications, drug delivery, and isolation of DNA and protein. Fungal pathogens that infect plants critically affect cost effectiveness, quality, and quantity of their production. They kill plant cells and/or cause plant stress. Plant fungal infections can originate from many sources such as infected soil, seeds, or crop debris causing diseases and quality losses around the world with billions of US dollars annually as costs of the associated productivity loss. This study focused on the application of the sponge-like protocol in protecting in vitro tissue cultures of plants against fungal pathogens. This can be useful for research purposes or may be developed to be introduced in field applications. Aspergillus flavus and Aspergillus niger infection in tissue culture of jojoba (Simmondsia chinensis (Link) Schn.) was used as a model to establish the employment of this protocol to control plant fungal diseases. The best conditions for A. flavus and A. niger ghosts production previously mapped by randomization experimental design (reduced Plackett-Burman experimental design) were used to prepare fungal ghosts. SDS, NaOH, NaHCO3, and H2O2 were used in their MIC (+1 level) or minimum growth concentration (MGC, -1 level) according to the determined optimal experimental design. The release of both of DNA and protein from the fungal cells was evaluated spectrophotometrically at 260nm and 280nm, respectively, as an indicator for cell loss of their cytoplasm. Fungal ghost cells were also examined by transmission electron microscopy. After confirming the preparation of high-quality fungal ghost cells, the same conditions were mimicked to control plant fungal infection. Jojoba grown in tissue culture was sprayed with fungal cells (about 103 CFU) as a control experiment or fungal cells followed by treatment with solution (a) represents the fungal ghost cells formation calculated critical concentration (FGCCC) of SDS, NaOH, and NaHCO3 and then treatment with solution (b) represents H2O2 FGCCC. The plant was examined on day 0 (plant grown before any infection or infection followed by treatment), day 5 (plant at day 5 after infection or infection followed by treatment), and day 10 (plant at day 10 after infection or infection followed by treatment). We observed fungal growth in case of control experiments at days 5 and 10 on the tissue culture medium, as well as plant, and the absence of any fungal growth in case of plant treated with FGCCC even after day 10. We recommend using this FGCCC in the form of chemical spraying formulation to treat the plants aiming to control different plant fungal infections in in vitro tissue culture systems or applied in field.

Lipases of germinating jojoba seeds efficiently hydrolyze triacylglycerols and wax esters and display wax ester-synthesizing activity.

BACKGROUND: Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds. RESULTS: During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds. CONCLUSIONS: In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Deciphering the molecular pathways of apoptosis using purified fractions from leaf extract of Basella alba through studying the regulation of apoptosis related genes.

Apoptosis plays a pivotal role in the exclusion of abnormal cells without any ruin of surrounding healthy cells. Generally, it occurs through an orderly and autonomously process which is controlled by proper function of various genes. Therefore, the current experiments detect the expression level/pattern of those genes to confirm the involvement of extrinsic and intrinsic pathway using Basella alba leaf (BAL). Several fractions after gel filtration chromatography of BAL extract have been pooled to evaluates its apoptosis induction potentiality on Ehrlich's Ascites Carcinoma (EAC) cells through conducting a number of bio-assays such as cell growth inhibition assay, fluorescence and optical microscopy, DNA fragmentation assay and gene expression analysis etc. The pooled fractions of BAL showed 12-56% inhibitory effect on EAC cell line at the concentration range of 25-400 µg/ml that was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. They also exhibited excellent cell growth inhibition at in vivo and in vitro condition when treated with 10, 20 and 40 mg/kg day. After administration of six consequent days, significant morphological features of apoptosis were observed in EAC cells under both fluorescence and optical microscope which was further supported by DNA fragmentation assay. The polymerase chain reaction amplification of bax, bcl-2 (B-cell lymphoma 2), p53, tumor necrosis factor-α, Fas, NF-kß (Nuclear factor-Kappa-B), PARP-1 (Poly (ADP-ribose) polymerase), Cyt-c cas-8, cas-9 and cas-3 revealed that the experimental sample able to induce apoptosis in both extrinsic and intrinsic pathways through altering the gene expression. The current findings suggest that sample from BAL occupy wonderful competence to induce cell apoptosis and become an ideal resource for cancer treatment.

Anthocyanin synthesis potential in betalain-producing Caryophyllales plants.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.