Maize resistance to witchweed through changes in strigolactone biosynthesis.

Maize (Zea mays) is a major staple crop in Africa, where its yield and the livelihood of millions are compromised by the parasitic witchweed Striga. Germination of Striga is induced by strigolactones exuded from maize roots into the rhizosphere. In a maize germplasm collection, we identified two strigolactones, zealactol and zealactonoic acid, which stimulate less Striga germination than the major maize strigolactone, zealactone. We then showed that a single cytochrome P450, ZmCYP706C37, catalyzes a series of oxidative steps in the maize-strigolactone biosynthetic pathway. Reduction in activity of this enzyme and two others involved in the pathway, ZmMAX1b and ZmCLAMT1, can change strigolactone composition and reduce Striga germination and infection. These results offer prospects for breeding Striga-resistant maize.

Developing Integrated Approaches for Testing and Assessment (IATAs) in order to support nanomaterial safety.

Due to the unique characteristics of nanomaterials (NM) there has been an increase in their use in nanomedicines and innovative medical devices (MD). Although large numbers of NMs have now been developed, comprehensive safety investigations are still lacking. Current gaps in understanding the potential mechanisms of NM-induced toxicity can make it challenging to determine the safety testing necessary to support inclusion of NMs in MD applications. This article provides guidance for implementation of pre-clinical tailored safety assessment strategies with the aim to increase the translation of NMs from bench development to clinical use. Integrated Approaches to Testing and Assessment (IATAs) are a key tool in developing these strategies. IATAs follow an iterative approach to answer a defined question in a specific regulatory context to guide the gathering of relevant information for safety assessment, including existing experimental data, integrated with in silico model predictions where available and appropriate, and/or experimental procedures and protocols for generating new data to fill gaps. This allows NM developers to work toward current guidelines and regulations, while taking NM specific considerations into account. Here, an example IATA for NMs with potential for direct blood contact was developed for the assessment of haemocompatibility. This example IATA brings together the current guidelines for NM safety assessment within a framework that can be used to guide information and data gathering for the safety assessment of intravenously injected NMs. Additionally, the decision framework underpinning this IATA has the potential to be adapted to other testing needs and regulatory contexts.

Oxygen surface exchange kinetics of Ba0.5Sr0.5Co0.8Fe0.2O3-δ.

The mechanism of oxygen exchange between the gas phase and Ba0.5Sr0.5Co0.8Fe0.2O3-δ oxide was evaluated by considering the inhomogeneity of the oxide surface. The applicability of existing models for the analysis of the oxygen exchange mechanism was considered. A new model with a dissociation step was suggested. The rate-determining steps of the oxygen exchange process were revealed under different experimental conditions. The change in the rate-determining step occurred at 600-650 °C. The probable cause was considered taking into account the parameter of nonequivalency of adsorption centers. A relationship between the oxygen isotope redistribution rates and the rates of the elementary steps in a "gas phase-solid oxide" system was revealed.

Detection of titanium particles in human liver and spleen and possible health implications.

BACKGROUND: Titanium dioxide (TiO2) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal studies. A health risk from nanosized TiO2 particle consumption could not be excluded then. RESULTS: Here we show the first quantification of both total titanium (Ti) and TiO2 particles in 15 post-mortem human livers and spleens. These low-level analyses were enabled by the use of fully validated (single particle) inductively coupled plasma high resolution mass spectrometry ((sp)ICP-HRMS) detection methods for total Ti and TiO2 particles. The presence of TiO2 in the particles in tissues was confirmed by Scanning Electron Microscopy with energy dispersive X-ray spectrometry. CONCLUSIONS: These results prove that TiO2 particles are present in human liver and spleen, with ≥24% of nanosize (< 100 nm). The levels are below the doses regarded as safe in animals, but half are above the dose that is deemed safe for liver damage in humans when taking into account several commonly applied uncertainty factors. With these new and unique human data, we remain with the conclusion that health risks due to oral exposure to TiO2 cannot be excluded.

Spatial Analysis of Temporal Changes in the Pandemic of Severe Cassava Mosaic Disease in Northwestern Tanzania.

To improve understanding of the dynamics of the cassava mosaic disease (CMD) pandemic front, geospatial approaches were applied to the analysis of 3 years' data obtained from a 2-by-2° (approximately 222-by-222 km) area of northwestern Tanzania. In total, 80 farmers' fields were assessed in each of 2009, 2010, and 2011, with 20 evenly distributed fields per 1-by-1° quadrant. CMD-associated variables (CMD incidence, CMD severity, vector-borne CMD infection, and vector abundance) increased in magnitude from 2009 to 2010 but showed little change from 2010 to 2011. Increases occurred primarily in the two westernmost quadrants of the study area. A pandemic "front" was defined by determining the values of CMD incidence and whitefly abundance where predicted disease gradients were greatest. The pandemic-associated virus (East African cassava mosaic virus-Uganda) and vector genotype (Bemisia tabaci sub-Saharan Africa 1-subgroup 1) were both present within the area bounded by the CMD incidence front but both also occurred ahead of the front. The average speed and direction of movement of the CMD incidence front (22.9 km/year; southeast) and whitefly abundance front (46.6 km/year; southeast) were calculated, and production losses due to CMD were estimated to range from US$4.3 million to 12.2 million.

Sulforaphane formation and bioaccessibility are more affected by steaming time than meal composition during in vitro digestion of broccoli.

Broccoli is a rich source of the glucosinolate glucoraphanin (GR). After hydrolysis of GR by the endogenous enzyme myrosinase, sulforaphane (SF) or sulforaphane nitrile (SFN) are produced, depending on environmental conditions. How the conversion of GR and bioaccessibility of released breakdown products are affected by steaming (raw, 1min, 2min and 3min steamed) and meal composition (protein or lipid addition) was studied with an in vitro digestion model (mouth, stomach, intestine, but not colonic digestion). The main formation of SF and SFN occurred during in vitro chewing. The contents of GR, SF and SFN did not change after further digestion, as the irreversible inactivated myrosinase under gastric conditions caused no further GR hydrolysis. SF concentrations were up to 10 times higher in raw and 1min steamed broccoli samples after digestion compared to longer-steamed broccoli. Protein or lipid addition had no influence on the formation and bioaccessibility of SF or SFN.

The Dynamics and Environmental Influence on Interactions Between Cassava Brown Streak Disease and the Whitefly, Bemisia tabaci.

Cassava brown streak disease (CBSD) is currently the most significant virus disease phenomenon affecting African agriculture. In this study, we report results from the most extensive set of field data so far presented for CBSD in Africa. From assessments of 515 farmers' plantings of cassava, incidence in the Coastal Zone of Tanzania (46.5% of plants; 87% of fields affected) was higher than in the Lake Zone (22%; 34%), but incidences for both zones were greater than previous published records. The whitefly vector, Bemisia tabaci, was more abundant in the Lake Zone than the Coastal Zone, the reverse of the situation reported previously, and increased B. tabaci abundance is driving CBSD spread in the Lake Zone. The altitudinal "ceiling" previously thought to restrict the occurrence of CBSD to regions <1,000 masl has been broken as a consequence of the greatly increased abundance of B. tabaci in mid-altitude areas. Among environmental variables analyzed, minimum temperature was the strongest determinant of CBSD incidence. B. tabaci in the Coastal and Lake Zones responded differently to environmental variables examined, highlighting the biological differences between B. tabaci genotypes occurring in these regions and the superior adaptation of B. tabaci in the Great Lakes region both to cassava and low temperature conditions. Regression analyses using multi-country data sets could be used to determine the potential environmental limits of CBSD. Approaches such as this offer potential for use in the development of predictive models for CBSD, which could strengthen country- and continent-level CBSD pandemic mitigation strategies.

Comparison of the chemical composition of three species of smartweed (genus Persicaria) with a focus on drimane sesquiterpenoids.

The genus Persicaria is known to include species accumulating drimane sesquiterpenoids, but a comparative analysis highlighting the compositional differences has not been done. In this study, the secondary metabolites of both flowers and leaves of Persicariahydropiper, Persicariamaculosa and Persicariaminor, three species which occur in the same habitat, were compared. Using gas chromatography-mass spectrometry (GC-MS) analysis of extracts, overall 21/29 identified compounds in extracts were sesquiterpenoids and 5/29 were drimanes. Polygodial was detected in all species, though not in every sample of P. maculosa. On average, P. hydropiper flowers contained about 6.2 mg g FW(-1) of polygodial, but P. minor flowers had 200-fold, and P. maculosa 100,000 fold lower concentrations. Comparatively, also other sesquiterpenes were much lower in those species, suggesting the fitness benefit to depend on either investing a lot or not at all in terpenoid-based secondary defences. For P. hydropiper, effects of flower and leaf development and headspace volatiles were analysed as well. The flower stage immediately after fertilisation was the one with the highest content of drimane sesquiterpenoids and leaves contained about 10-fold less of these compounds compared to flowers. The headspace of P. hydropiper contained 8 compounds: one monoterpene, one alkyl aldehyde and six sesquiterpenes, but none were drimanes. The potential ecological significance of the presence or absence of drimane sesquiterpenoids and other metabolites for these plant species are discussed.

Bioaccessibility of vitamin A, vitamin C and folic acid from dietary supplements, fortified food and infant formula.

In the Netherlands, vitamin intake occurs mainly via food and for some vitamins also via fortified food. In addition, some people take dietary supplements. Information on the bioavailability of vitamins is important for a good estimation of the actual exposure to vitamins. Furthermore, for a reliable intake estimation, it is important to know the accurateness of the claimed vitamin concentration on the product label. In the current study, the amount of vitamin A, vitamin C, and folic acid in different products and their maximum bioavailability (bioaccessibility) were investigated. In about half of the products, the amount of vitamins significantly deviated from the declared amounts. The vitamin bioaccessibility ranged from <1% to 100%. When assessing the dietary intake exposure of vitamins, it is important to take into account both the possible deviation from the declared level and (the variability of) the bioaccessibility of the vitamin in the products.

ABA-deficiency results in reduced plant and fruit size in tomato.

Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.

Detection of diseased plants by analysis of volatile organic compound emission.

This review focuses on the detection of diseased plants by analysis of volatile organic compound (VOC) emissions. It includes an overview of studies that report on the impact of infectious and noninfectious diseases on these emissions and discusses the specificity of disease-induced emissions. The review also provides an overview of processes that affect the gas balance of plant volatiles, including their loss processes. These processes are considered as important because they contribute to the time-dynamic concentration profiles of plant-emitted volatiles. In addition, we describe the most popular techniques currently in use to measure volatiles emitted from plants, with emphasis on agricultural application. Dynamic sampling coupled with gas chromatography and followed by an appropriate detector is considered as the most appropriate method for application in agriculture. It is recommended to evaluate the state-of-the-art in the fields concerned with this method and to explore the development of a new instrument based on the specific needs for application in agricultural practice. However, to apply such an instrument in agriculture remains a challenge, mainly due to high costs.

Comparing the regional epidemiology of the cassava mosaic and cassava brown streak virus pandemics in Africa.

The rapid geographical expansion of the cassava mosaic disease (CMD) pandemic, caused by cassava mosaic geminiviruses, has devastated cassava crops in 12 countries of East and Central Africa since the late 1980s. Region-level surveys have revealed a continuing pattern of annual spread westward and southward along a contiguous 'front'. More recently, outbreaks of cassava brown streak disease (CBSD) were reported from Uganda and other parts of East Africa that had been hitherto unaffected by the disease. Recent survey data reveal several significant contrasts between the regional epidemiology of these two pandemics: (i) severe CMD radiates out from an initial centre of origin, whilst CBSD seems to be spreading from independent 'hot-spots'; (ii) the severe CMD pandemic has arisen from recombination and synergy between virus species, whilst the CBSD pandemic seems to be a 'new encounter' situation between host and pathogen; (iii) CMD pandemic spread has been tightly linked with the appearance of super-abundant Bemisia tabaci whitefly vector populations, in contrast to CBSD, where outbreaks have occurred 3-12 years after whitefly population increases; (iv) the CMGs causing CMD are transmitted in a persistent manner, whilst the two cassava brown streak viruses appear to be semi-persistently transmitted; and (v) different patterns of symptom expression mean that phytosanitary measures could be implemented easily for CMD but have limited effectiveness, whereas similar measures are difficult to apply for CBSD but are potentially very effective. An important similarity between the pandemics is that the viruses occurring in pandemic-affected areas are also found elsewhere, indicating that contrary to earlier published conclusions, the viruses per se are unlikely to be the key factors driving the two pandemics. A diagrammatic representation illustrates the temporal relationship between B. tabaci abundance and changing incidences of both CMD and CBSD in the Great Lakes region. This emphasizes the pivotal role played by the vector in both pandemics and the urgent need to identify effective and sustainable strategies for controlling whiteflies on cassava.

Trichome dynamics and artemisinin accumulation during development and senescence of Artemisia annua leaves.

Artemisinin is a sesquiterpene lactone endoperoxide and an important antimalarial drug produced in Artemisia annua. To unravel the diverse processes determining artemisinin yield in A. annua crops, artemisinin accumulation during the development of individual leaves was studied in two field experiments. During the life cycle of a leaf, artemisinin was always present. Quantities were low at leaf appearance and increased steadily. In leaves studied until after senescence, maximum quantities and concentrations were achieved after the leaf had turned brown. The total quantity of possible artemisinin precursors per leaf (dihydroartemisinic acid and other upstream precursors) was highest early in the leaf cycle when the leaf was still expanding. Dihydroartemisinic acid was more abundant than the other compounds and its quantity declined during leaf development whereas that of artemisinin increased. Dihydroartemisinic acid was not converted directly into artemisinin, because on a per leaf basis the decline in molar quantity of precursors in the earliest formed leaves was not compensated for by a simultaneous increase in artemisinin. Our results suggest that a (putative) intermediate such as dihydroartemisinic acid hydroperoxide temporarily may have accumulated in considerable quantities. The number of mature, capitate trichomes on the adaxial leaf side increased after leaf appearance until the end of leaf expansion, and then decreased, probably due to collapse of trichomes. Artemisinin production thus (also) occurred when trichomes were collapsing. Later formed leaves achieved higher concentrations of artemisinin than earlier formed leaves, because of a higher trichome density and a higher capacity per trichome.

Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.

An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the--so far unknown--amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid.

Biotransformation of limonene by bacteria, fungi, yeasts, and plants.

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.

Role of cucurbitacin C in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis sativus L.).

Cucurbitacins are bitter triterpenoid compounds that are toxic to most organisms and occur widely in wild and cultivated Cucurbitaceae. The only cucurbitacin identified in Cucumis sativus is cucurbitacin C. The bitter taste of cucumber has been correlated with resistance to the spider mite Tetranychus urticae, but a quantitative relationship has not been established. We determined the spider mite resistance and cucurbitacin C content in the dihaploid progeny derived from the F1 generation of a cross between a bitter, spider-mite-resistant cucumber line and a bitter-free, spider-mite-susceptible line. The ratio of the number of bitter to bitter-free dihaploids conformed to the expected 1:1 ratio, based on a monogenic segregation pattern. Genetic analysis ascribed 69% of the variance of the difference in spider mite survival rate to the bitterness locus. Within the group of bitter dihaploids, cucurbitacin C content was significantly correlated with spider mite resistance. Thus, a quantitative relationship between cucurbitacin C content and spider mite resistance could be established.

Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranoside.

Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non-volatile S-linalyl-beta-D-glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.

Germacrenes from fresh costus roots.

Four germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots (Saussurea lappa). The structures of (+)-germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of these compounds yields (-)-beta-elemene, (-)-elema-1,3,11(13)-trien-12-ol, (-)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien-12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the germacrenes yields selinene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are artefacts.

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.

The sesquiterpenoid artemisinin, isolated these from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight.

Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis.

Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates beta-elemene with a modest degree of enantioselectivity.