Thunbergia Alata Research Articles

FORMULATION AND ASSESSMENT OF HERBAL EMULGELS IN THE MANAGEMENT OF ACNE: IN VITRO AND IN VIVO INVESTIGATIONS

Objective: The main objective of the current research was to prepare herbal emulgel and analyze the effect of herbal formulation in the treatment of acne. Methods: The plants Tabernaemontana coronaria and Thunbergia alata were selected for the study because of folklore for their medicinal values. The T. coronaria and T. alata test extracts were prepared by soxhlet extraction procedure and subjected to physico-chemical evaluation. The formulated herbal emulgels prepared by dispersion technique were investigated for anti-acne properties by in vitro and in vivo methods. The prepared emulgel formulations were assessed for parameters like viscosity, spreadability, pH, content uniformity, stickiness, zeta potential, particle size, surface morphology, and in vitro diffusion studies. Results: The physico-chemical evaluation of herbal gel revealed that emulgel appeared light green in colour, opaque, and odourless with smooth texture. The emulgels of both the test extracts showed no stickiness, and revealed pH ranging from 5.467±0.13 to 5.889±0.1. When the shear rate was increased, there was a decrease in the viscosity of the test emulgels, with good extrudability. The content uniformity of F5 emulgel for T. coronaria and T. alatawas 99%, and spreadibilty was more with F7 formulation of T. coronaria and F6 formulation of T. alata, respectively. In the stability testing studies, amongst all the formulations prepared, F5 was found to be stable upon storage for six months. In vitro studies, F5 formulation of both the test extracts had a remarkable zone of inhibition; whereas F5 formulation treated histopathological sections in in vivo investigation displayed a decline in the overall damage induced by Propionibacterium acnes. The results showed no statistical significant difference for measurement of zone of inhibition and histopthological studies between the test formulations and standard drug. Conclusion: The study concludes that both herbal formulations were promising agents for the treatment of acne vulgaris.

Black-eyed Susan vine (Thunbergia alata): chemical and antifungal potential evaluation of an invasive plant species in Colombia

Thunbergia alata (Black-eyed Susan) is a plant species from East Africa and grows in tropical regions worldwide, including Colombia, being considered an invasive species in some countries. Even though it is used by local communities to treat several illnesses, including malaria, there are few biological and chemical studies on T. alata. Therefore, this study aimed to explore the chemical composition and in vitro antimycotic (against Candida strains) activity of T. alata crude ethanolic stem and leaf extracts. This was achieved via phytochemical analyses and chromatographic profiling (HPTLC, UPLC-DAD, and LC-MS). A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against selected Candida strains. Results showed the presence of saponins, terpenes, and flavonoids in the T. alata crude ethanolic stem and leaf extracts, although rutin and chlorogenic acid were the main components of stem and leaf extracts, respectively. Concerning biological assays, T. alata leaf extracts demonstrated moderate antimycotic activity, with MIC values between 5:00 mg mL-1 and 1:25 mg mL-1 against C. albicans and C. auris strains. The results indicate that T. alata extracts, including some of their major identified compounds, possess promising antifungal properties against two significant microorganisms.

MALDI-MS screening of microbial colonies with isomer resolution to select fatty acid desaturase variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid MS assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

Ecological, phytosanitary, and agronomic aspects of target weeds for biological control studies in Antioquia, Colombia

Biological control as part of integrated weed management represents an emerging and promising method to minimize the use and impact of herbicides on the environment. The objective of this research was to determine the ecological, phytosanitary, and agronomic status of weeds associated with crops in Antioquia, Colombia, and their potential to be chosen as candidates for biological control studies using pathogenic fungi. The research was carried out through a sampling of weeds in 35 agricultural farms, distributed in 11 municipalities and 10 different crops, on which population ecological variables such as frequency and dominance were evaluated; phytosanitary variables such as incidence and severity of foliar fungal symptoms; as well as some biological and anthropogenic aspects of these, through interviews with the farmers of each farm. The integration of these variables was proposed as an indicator to order and classify the plants according to their greater suitability to be subjected to biocontrol studies. A diversity of 75 weed species was found, with the Poaceae and Asteraceae families standing out, which together represented 43% of the population. 76% of this population presented disease symptoms, presumably of fungal origin, with the severity of up to 10% of the affected leaf area. In 22% of the species agronomic management difficulties greater than 50% were detected on a scale of 1 to 10. We indicate the following twelve weed species as candidates for future biological control studies through phytopathogenic fungi: Rumex crispus L., Digitaria horizontalis (Willd), Persicaria nepalensis (Meisn.) Miyabe, Thunbergia alata Bojer ex Sims, Melinis minutiflora Beauv., Paspalum paniculatum L., Pennisetum clandestinum Hochst. ex Chiov., Pteridium aquilinum (L.) Kunth, Sonchus oleraceus L., Erigeron bonariensis L., Bidens pilosa L., and Artemisia absinthium L. This is the first report of its kind in Colombia.

Regioselectivity of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase by molecular dynamics.

The plant acyl-acyl carrier protein (ACP) desaturases are a family of soluble enzymes that convert saturated fatty-acyl ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction. These enzymes play a key role in biosynthesis of mono-unsaturated fatty acids in plants. The archetype of this group is the Δ9-18:0-ACP desaturase that introduces a cis double bond between carbons 9 and 10 to produce oleoyl-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). ACPs are central proteins in the fatty acid biosynthesis that deliver the substrate to the desaturase. They have been reported to show a varying degree of local dynamics and structural variability depending on the substrate length. It has been suggested that the substrate specific change in structure and dynamics is crucial for the decreasing enzymatic activity in the case of the Δ9-Desaturase for chains length shorter than 18. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position carbons 6 and 7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Air temperature during cutting propagation of cold-intermediate and -sensitive crops can be reduced if root-zone heating is provided

Asexual propagation of herbaceous ornamentals occurs from late winter to early spring coincides with seasonally low daily light integrals (DLIs) and average daily temperatures (ADT) outdoors. Supplemental lighting and air and root-zone heating (RZH) can be used to promote rapid callus induction and rooting of cuttings. Due to the range of greenhouse crop base temperatures (cold-tolerant, cold-intermediate, and cold-sensitive), one strategy is to reduce the air ADT of certain crops while using RZH as a potential energy saving strategy. Therefore, the objective of this study was to quantify how reduced air ADT and increased RZH temperatures influence root initiation and development of cold-intermediate and-sensitive herbaceous annual and perennial crops propagated by shoot-tip cuttings. Unrooted vegetative cuttings of angelonia ‘Archangel Raspberry’ (Angelonia angustifolia), lantana ‘Landmark Sunrise Rose Improved’ (Lantana camara), New Guinea impatiens ‘Clockwork White Improved’ (Impatiens hawkeri), scaevola ‘Scalora Brilliant’ (Scaevola aemula), thunbergia ‘Sunny Lemon Star’ (Thunbergia alata), verbena ‘Cadet Upright Red’ (Verbena × hybrida), geranium ‘Moxie! Deep Rose’ (Pelargonium interspecific), Kwik Kombos ‘Pink Lemonade Mix’ [petunia ‘Dekko Star Rose’ (Petunia × hybrida), lobelia ‘Techno Heat Light Blue Improved’ (Lobelia erinus), and calibrachoa ‘Callie Yellow Improved’ (Calibrachoa × hybrida)] and portulaca ‘Cupcake Carrot’ and ‘Colorblast Lemon Twist’ (Portulaca grandiflora) were inserted into a soilless medium and placed in a greenhouse with an air ADT of 21°C and RZH set point of 24°C to callus. After 6 d, trays of cuttings were placed between two greenhouses with an air ADT of 16 or 21°C and among 3 benches which provided RZH set points of 21, 24, or 27°C. After 14 or 16 d post-callus, measurements were collected for all genera and geranium and portulaca ‘Colorblast Lemon Twist’ were transplanted into a common environment. The air ADT and RZH individually influenced stem length of angelonia, lantana, kwik kombos, portulaca, thunbergia, and verbena, and interacted to influence angelonia and lantana. Under an air ADT of 21°C, angelonia was 30%-45% taller than an air ADT of 16°C. Stem length of geranium, New Guinea impatiens, and scaevola were not influenced by air ADT or RZH. With an RZH set point of 21°C, the root dry mass of thunbergia was 72% greater under an air ADT of 21 than 16°C. Lantana shoot dry mass was 23% smaller under an air ADT of 16°C and RZH of 21°C compared to an RZH of 24°C. Finished plants of geranium and portulaca were not influenced by air ADT or RZH and were comparable in size and date of marketability between treatments. Due to little to no differences in crop timing or quality, the majority of the crops studied can be propagated with an air ADT and RZH of 16 and 24°C, respectively, without negative effects during or after propagation.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Abstract Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Phytopathogenic fungi with potential as biocontrol agents for weeds of importance in crops of Antioquia, Colombia

BackgroundUse of phytopathogenic fungi for the biocontrol of weeds represents a promising path in the search for new management alternatives that allow reducing negative effects on the environment and the generation of biotypes having resistance to herbicides. The first step in developing weed biological control programs is to determine the plants and their natural enemies with the highest affinity and potential to achieve effective biocontrol. The objective of the present study was to evaluate the phytopathogenic potential of fungal isolates on four economically important weeds including: Rumex crispus L., Digitaria horizontalis Willd, Persicaria nepalensis (Meisn.) Miyabe, and Thunbergia alata Bojer ex Sims, as a possible biocontrol agent.ResultsMorphological and molecular identification of nine phytopathogenic isolates of weeds was achieved, according to the sequencing of the ITS, β-Tub2, and TEF1-α regions. Pathogenicity of the following species on original hosts was confirmed: Colletotrichum cigarro, Epicoccum draconis, and Didymella rumicicola on R. crispus; Bipolaris sp., on D. horizontalis; Bipolaris zeicola, Phialemoniopsis curvata, and Stemphylium beticola on P. nepalensis and, Alternaria thunbergiae and Nigrospora sphaerica on T. alata. These could be, fairly, considered the first worldwide reports of such interactions, except for A. thunbergiae and Bipolaris sp. The most virulent interactions according to the AUDPC value corresponded to (R. crispus × D. rumicicola), (D. horizontalis × Bipolaris sp.), (P. nepalensis × S. beticola) and (T. alata × A. thunbergiae), with an incidence of 100%.ConclusionThese strains were proposed for future research as potential biocontrol agents, which represented a great resource for the possible generation of new bio-herbicides.

Chemical profiling of polyphenols in Thunbergia alata and in silico virtual screening of their antiviral activities against COVID-19

A comprehensive analysis of the polyphenols constituents found in Thunbergia alata leaves using (UPLC-Triple TOF-MS/MS) and natural polyphenols' antiviral properties have motivated us to perform in silico binding affinity study of flavonoids and coumarins identified in T. alata leaves extract against different proteins of SARS-COV-2; (Mpro) main protease (PDB ID: 6LU7) and 2′-Omethyltransferase (PDB ID: 6wkq) using a co-crystalized ligands N3 and Sinefungin respectively. 30 comp.s were tentatively identified in T. alata leaves extract by UPLC-Triple TOF-MS/MS and characterized as 8-phenolic acids; 12-flavonoids; 3-coumarins and remain comp.s unidentified. In silico virtual screening of both flavonoids and coumarins as the most common classes of polyphenols; Flavonoids have the best potential to act as COVID-19 Mpro & MTase inhibitors; especially Kaempferol-3-O-(6-p-coumaroyl)-glucoside; Comp. (17) which gave very best docking score{ -9.2 kcal/mol} (docking energy score of N3 -7.6 kcal/mol). It bind by nine hydrogen bonds (with TRY54, CYS145, GLN192, PHE140, LEU141, THR26, ARG188 and ASN142), and Pi-Sulfur interaction (with MET165), hydrophobic interactions via five Pi-Alkyl bond (with MET49, CYS145 and PRO168) and also scored energy binding -10.2 (docking energy score of Sinefungin -7.8 kcal/mol); formed H-bond with ASN6841, ASN6899, CYS6913, LYS6968, GLY6869, GLY6871, ASP6928, ASN6996 and TRY6930. Moreover, 17 binds with LYS6844, LYS6935, LEU6898 and CYS6913 via hydrophobic interactions. While coumarins in comparison with the current flavonoids had less energy binding score. However, further research is important to investigate their potential medicine value.

Efficiency Evaluation and Usages of Thunbergia alata, Thunbergia erecta and their Combination

Thunbergiaalata and Thunbergiaerecta have been mostly used for ornamentation but have interesting other uses. However, toxicity and phytochemical studies of these plants are lacking. Therefore, phytochemicals of the two species were investigated by gas chromatography-mass spectrometry, cytotoxicity and genotoxicity were tested via 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide and comet assays in normal human peripheral blood mononuclear cells. Then, the extracts of the two species and their combination were applied to peripheral blood mononuclear cells poisoned with rice whisky and bathroom cleaner as toxic models in everyday life. The results showed that the major phytochemicals were 12.36 % and 24.90 % phytol in ethanol extracts of Thunbergia alata and Thunbergia erecta and 46.29 % and 43.47 % oleamide in hexane extracts of Thunbergia alata and Thunbergia erecta, respectively. Half-maximal inhibitory concentration values, which indicate toxicity in cells, were not detected for either species, but at the deoxyribonucleic acid level, the extracts induced significant deoxyribonucleic acid damage, shown by high Olive Tail Moment values in peripheral blood mononuclear cells (p<0.05). Biological activity of the extracts revealed by higher cell viability percentages and lower Olive Tail Moment values in the treatments (poisoned peripheral blood mononuclear cells treated with plant extracts) than the controls (poisoned peripheral blood mononuclear cells). Taking all the results together, Thunbergia alata and Thunbergia erecta extracts and their combination can be applied for many benefits in humans following properties that have been previously used and their phytochemicals but have limitations of doses.

First record ofAlternaria thunbergiaeonThunbergia alatain Europe

Thunbergia alata, often known as black-eyed Susan vine, is used in UK gardens as an annual climber in bedding schemes and hanging baskets. Flowers are typically shades of yellow, orange and red with a black centre. Plants germinated onsite …

Thunbergia alata inhibits inflammatory responses through the inactivation of ERK and STAT3 in macrophages.

Thunbergiaalata (Acanthaceae) has been used traditionally to treat various inflammatory diseases such as fever, cough and diarrhea in East African countries including Uganda and Kenya. However, systemic studies elucidating the anti-inflammatory effects and precise mechanisms of action of T.alata have not been conducted, to the best of our knowledge. To address these concerns, we explored the anti-inflammatory effects of a methanol extract of T.alata(MTA) in macrophages. Non-cytotoxic concentrations of MTA(≤300µg/ml) inhibited nitric oxide(NO) production in lipopolysaccharide(LPS)‑stimulated RAW264.7 macrophages by transcriptional regulation of inducible NO synthase in a dose-dependent manner. The expression of cyclooxygenase-2, the enzyme responsible for the production of prostaglandinE2, was unchanged by MTA at the mRNA and protein levels. MTA treatment inhibited interleukin(IL)-6 production and decreased the mRNA expression of pro‑inflammatory cytokines, including IL-6 and IL-1β. Tumor necrosis factor-α production and mRNA expression were not regulated by MTA treatment. The decreased production of inflammatory mediators by MTA was followed by the reduced phosphorylation of extracellular signal‑regulated kinase(ERK) and signal transducer and activator of transcription3(STAT3). MTA treatment had no effect on activity of other mitogen‑activated protein kinases(MAPKs), p38, c-Jun N-terminal kinase(JNK), and nuclear factor-κB(NF-κB). These results indicate that MTA selectively inhibits the excessive production of inflammatory mediators in LPS-stimulated murine macrophages by reducing the activity of ERK and STAT3, suggesting that MTA plays an important inhibitory role in the modulation of severe inflammation.

Acanthaceae da Marambaia, Estado do Rio de Janeiro, Brasil

RESUMO Este trabalho consiste do tratamento taxonômico das Acanthaceae Juss. na Marambaia, Estado do Rio de Janeiro, e sua distribuição nas diferentes formações vegetais locais. Foram constatados cinco gêneros e nove espécies encontradas nos manguezais, na restinga e na floresta ombrófila, além de áreas urbanizadas. Thunbergia alata é introduzida e amplamente naturalizada no Brasil inteiro e Ruellia blechum é uma espécie neotropical também naturalizada; as demais espécies (Avicennia schaueriana, Aphelandra longiflora, Justicia brasiliana, J. cydoniifolia, J. plumbaginifolia, Ruellia solitaria e Schaueria litoralis) são nativas. São apresentadas chave de identificação, descrições, ilustrações, comentários taxonômicos e a ocorrência nas formações vegetais locais, em outras restingas fluminenses e no Brasil. As espécies de Acanthaceae da Marambaia são recursos importantes para diferentes grupos de polinizadores e reúnem espécies típicas (Justicia brasiliana, J. cydoniifolia) e exclusivas (Schaueria litoralis) das restingas; essas espécies, juntamente com Avicennia schaueriana, são ameaçadas de extinção na flora fluminense.

SENSITIVITY PATTERNS OF SOME FLOWERING PLANTS AGAINST SALMONELLA TYPHI AND PSEUDOMONAS AERUGINOSA

The medicinal plants have been collected form wild resources and only some species used in larger quantities are cultivated systematically. Many medicinal plants, which were ignored in the past years, but the number of plants still formed in the wild is progressively declining the medicinal value with this situation to acquiring from the wild sources in more important recent trends bening. Reported in cresingly the antimicrobial properties of medicinal plants from different parts of the world .On this basis, the present investigation was focused on the screening of antimicrobial properties ofAsystasia indica Asystasia gangetica Thunbergia alata on selected Pathogenic bacteriaThe main objectives of this work use as follow To Screen the antimicrobial properties of these plants on selected micro organisms.To determine the effectiveness of inhibition on microbes by comparing its activity with known antibiotics.

Selenopeptides and elemental selenium in Thunbergia alata after exposure to selenite: quantification method for elemental selenium.

Three month old Thunbergia alata were exposed for 13 days to 10 μM selenite to determine the biotransformation of selenite in their roots. Selenium in formic acid extracts (80 ± 3%) was present as selenopeptides with Se-S bonds and selenium-PC complexes (selenocysteinyl-2-3-dihydroxypropionyl-glutathione, seleno-phytochelatin2, seleno-di-glutathione). An analytical method using HPLC-ICPMS to detect and quantify elemental selenium in roots of T. alata plants using sodium sulfite to quantitatively transform elemental selenium to selenosulfate was also developed. Elemental selenium was determined as 18 ± 4% of the total selenium in the roots which was equivalent to the selenium not extracted using formic acid extraction. The results are in an agreement with the XAS measurements of the exposed roots which showed no occurrence of selenite or selenate but a mixture of selenocysteine and elemental selenium.

Selenite enhances arsenate toxicity in Thunbergia alata

Environmental context. Arsenic and selenium are two elements ubiquitously distributed in our environment. While selenium is not known to be essential to plants, it is certainly toxic as is arsenic. On the other hand selenium is known to be essential for mammals and an increase in selenium concentration in plants when used as feed or food may be beneficial for mammals in regions of low selenium concentration. The wide distribution of these two elements in the soil might result in elevated co-exposure of plants. So far no interactions of those elements in plants have ever been studied, although this might be mandatory when nutritionists would like to increase the selenium concentrations in crops, which are already challenged by elevated levels of arsenic. Abstract. The suspected antagonism between selenite and arsenite in various mammalian species gave reason to investigate the effect of selenite on arsenate toxicity in higher plants using Thunbergia alata as a model plant. Here, we present a study involving (1) toxicity tests, (2) uptake and translocation experiments as well as (3) selenium–sulfur–arsenic speciation analysis by HPLC-ESMS-ICP-MS (oxygen) of selected plant tissues. The EC50 (effective concentration that inhibits growth by 50%) values, as a result of the toxicity studies, showed that selenite co-exposure enhances arsenate toxicity. The detection of two selenium species, SeII-PC2 complex and Se-cysteinylserine glutathione, suggested that the increased toxicity symptoms might have been a result of the competition of SeII with AsIII for sulfhydryl groups (crucial for arsenite detoxification in plant cells) as binding partners.

Stability of arsenic peptides in plant extracts: off-line versus on-line parallel elemental and molecular mass spectrometric detection for liquid chromatographic separation

The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin (As(III)PC) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2% of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83% of arsenic was bound to peptides, mainly as As(III)PC(3) and (GS)As(III)PC(2). Key analytical factors were identified which destabilise the As(III)PCs. The low pH of the mobile phase (0.1% formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As(III)(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although As(III)PC(3) showed a ten times higher half-life (23 h) in a plant extract, the pre-cleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90% of an As(III)PC(3) complex and smaller free peptides such as PC(2) and PC(3) can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of As(III)PC(3) in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As(v) L(-1) was found when the fresh plant was stored for 21 days at 193 K.

What limits production of unusual monoenoic fatty acids in transgenic plants?

Unusual monounsaturated fatty acids are major constituents (greater than 80%) in seeds of Coriandrum sativum L. (coriander) and Thunbergia alata Bojer, as well as in glandular trichomes (greater than 80% derived products) of Pelargonium x hortorum (geranium). These diverged fatty acid structures are produced via distinct plastidial acyl-acyl carrier protein (ACP) desaturases. When expressed in Arabidopsis thaliana (L.) Heynh. under strong seed-specific promoters the unusual acyl-ACP desaturases resulted in accumulation of unusual monoene fatty acids at 1-15% of seed fatty acid mass. In this study, we have examined several factors that potentially limit higher production of unusual monoenes in transgenic oilseeds. (i) Immunoblots indicated that the introduced desaturases were expressed at levels equivalent to or higher than the endogenous delta9 18:0-ACP desaturase. However, the level of unusual fatty acid produced in transgenic plants was not correlated with the level of desaturase expression. (ii) The unusual desaturases were expressed in several backgrounds, including antisense 18:0-ACP desaturase plants, in fab1 mutants, and co-expressed with specialized ACP or ferredoxin isoforms. None of these experiments led to high production of expected products. (iii) No evidence was found for degradation of the unusual fatty acids during seed development. (iv) Petroselinic acid added to developing seeds was incorporated into triacylglycerol as readily as oleic acid, suggesting no major barriers to its metabolism by enzymes of glycerolipid assembly. (v) In vitro and in situ assay of acyl-ACP desaturases revealed a large discrepancy of activity when comparing unusual acyl-ACP desaturases with the endogenous delta9 18:0-ACP desaturase. The combined results, coupled with the sensitivity of acyl-ACP desaturase activity to centrifugation and low salt or detergent suggests low production of unusual monoenes in transgenic plants may be due to the lack of, or incorrect assemble of, a necessary multi-component enzyme association.

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

Purification and Identification of a Rhabdovirus from Thunbergia alata

A subgroup 2 rhabdovirus was isolated in south-east Queensland from black-eyed Susan (Thunbergia alata) with symptoms of vein yellowing, vein clearing and leaf distortion. Bacilliform particles accumulated in the perinuclear space of infected plants and measured 69 +/- 7 x 161 +/- 8 nm in unfixed preparations. The virus was not transmitted mechanically. Purified preparations of the Thunbergia alata rhabdovirus (TaRV) contained four major proteins with molecular weights of 80 kD, 48 kD, 40 kD and 35 kD, similar to those of datura yellow vein virus (DYVV), a newly described rhabdovirus from Australia. The 80 kD protein was identified as the viral glycoprotein. In immunoblots, the two largest proteins of TaRV reacted strongly with antiserum to DYVV, but were serologically distinct from sonchus yellow net, cereal chlorotic mottle, potato yellow dwarf and lettuce necrotic yellows viruses. TaRV is considered to be a strain of DYVV.