Dioxygenase from Aspergillus fumigatus MC8: molecular modelling and in silico studies on enzyme–substrate interactions

Flavoenzymes have been extensively studied for their structural and mechanistic properties because they find potential application as industrial biocatalysts. They are attractive for biocatalysis because of the selectivity, controllability and efficiency of their reactions. Some of these enzymes catalyse the oxidative modification of protein substrates. Among them oxygenases (monoxoygenases and dioxygenases) are of special interest because they are highly entantio as well as regio-selective and can be used for oxyfunctionalisation. Dioxygenase enzymes catalyse oxygenation reactions in which both di-oxygen atoms are incorporated into the product. A dioxygenase enzyme purified from Aspergillus fumigatus MC8 was subjected to protein digestion followed by peptide sequencing. The sequence analysis of the peptide fragments resulted in identifying its match with that of an extracellular dioxygenase sequence from the same species of fungus existing in the protein database. The sequence was submitted to protein homology/analogy recognition engine online server for homology modelling and the 3D structure was predicted. Subsequently, the in silico studies of the enzyme–substrate (protein–ligand) interaction were carried out by using the method of molecular docking simulations wherein the modelled dioxygenase enzyme (protein) was docked with the substrates (ligands), catechin and epicatechin.     

Searching Responses of a Hunting Spider to Cues Associated with Lepidopteran Eggs

Cheiracanthium inclusum (Hentz), a hunting spider, feeds on eggs of Helicoverpa zea (Boddie) and other moths. This study investigated whether C. inclusum can use chemical compounds present in H. zea scales and egg residues as kairomones to find these non-motile prey items. In a series of no- choice tests, spiders were presented with a piece of florist paper containing scales alone, scales + egg residues, or untreated controls. Next, spiders were presented with solvent extracts of either scales or eggs. Polar and non-polar solvents were used in the extractions. Contact with scales alone, scales + egg residues, and non-polar solvent extracts of both scales and eggs resulted in retention and/or induction of local searching behavior. Extracts made with polar solvents induced no apparent response, indicating that the chemostimulatory compounds are lipophilic. These results show that C. inclusum responds to kairomones left by ovipositing H. zea and use these chemical cues to detect and locate H. zea eggs.     

REGULATION OF AMYLASE,CELLULASE AND CHITINASE SECRETION IN THE DIGESTIVE TRACT OF THE TWO‐SPOTTED FIELD CRICKET,Gryllus bimaculatus

The secretion of amylase and cellulase in Gryllus bimaculatus is determined by increased food intake, whereby shortly after molting food consumption increases. About half of the standing amylase concentration (activity) in the endothelial cells can be secreted within 30 min. The peak of amylase and cellulase secretion that occurs in the photophase is related to the feeding peak in the previous scotophase. The secretion of chitinase on the other hand is primarily controlled by the molting cycle. Only amylase secretion was affected by calcium in the incubation medium, suggesting an apocrine release mechanism. Refeeding experiments (after 5 days without food) suggest that the release of amylase in response to a nutrient in the lumen (glucose) is not due to simple stimulation of exocytosis, but rather a stimulation of synthesis.     

Fruit cuticle lipid composition and water loss in a diverse collection of pepper (Capsicum)

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical‐compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14‐fold range for total wax amounts and a more than 16‐fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n‐alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper.     

Hyperaccumulators of metal and metalloid trace elements: Facts and fiction

Background

Plants that accumulate metal and metalloid trace elements to extraordinarily high concentrations in their living biomass have inspired much research worldwide during the last decades. Hyperaccumulators have been recorded and experimentally confirmed for elements such as nickel, zinc, cadmium, manganese, arsenic and selenium. However, to date, hyperaccumulation of lead, copper, cobalt, chromium and thallium remain largely unconfirmed. Recent uses of the term in relation to rare-earth elements require critical evaluation.

Scope

Since the mid-1970s the term ‘hyperaccumulator’ has been used millions of times by thousands of people, with varying degrees of precision, aptness and understanding that have not always corresponded with the views of the originators of the terminology and of the present authors. There is therefore a need to clarify the circumstances in which the term ‘hyperaccumulator’ is appropriate and to set out the conditions that should be met when the terms are used. We outline here the main considerations for establishing metal or metalloid hyperaccumulation status of plants, (re)define some of the terminology and note potential pitfalls.

Conclusions

Unambiguous communication will require the international scientific community to adopt standard terminology and methods for confirming the reliability of analytical data in relation to metal and metalloid hyperaccumulators.     

Partitioning of belowground C in young sugar maple forest

Background and aims

Trees allocate a high proportion of assimilated carbon belowground, but the partitioning of that C among ecosystem components is poorly understood thereby limiting our ability to predict responses of forest C dynamics to global change drivers.

Methods

We labeled sugar maple saplings in natural forest with a pulse of photosynthetic ¹³C in late summer and traced the pulse over the following 3 years. We quantified the fate of belowground carbon by measuring ¹³C enrichment of roots, rhizosphere soil, soil respiration, soil aggregates and microbial biomass.

Results

The pulse of ¹³C contributed strongly to root and rhizosphere respiration for over a year, and respiration comprised about 75 % of total belowground C allocation (TBCA) in the first year. We estimate that rhizosphere carbon flux (RCF) during the dormant season comprises at least 6 % of TBCA. After 3 years, 3.8 % of the C allocated belowground was recovered in soil organic matter, mostly in water-stable aggregates.

Conclusions

A pulse of carbon allocated belowground in temperate forest supplies root respiration, root growth and RCF throughout the following year and a small proportion becomes stabilized in soil aggregates.     

Biochar’s effect on crop productivity and the dependence on experimental conditions—a meta-analysis of literature data

Background and aims

For the last decade, there has been an increasing global interest in using biochar to mitigate climate change by storing carbon in soil. However, there is a lack of detailed knowledge on the impact of biochar on the crop productivity in different agricultural systems. The objective of this study was to quantify the effect of biochar soil amendment (BSA) on crop productivity and to analyze the dependence of responses on experimental conditions.

Methods

A weighted meta-analysis was conducted based on data from 103 studies published up to April, 2013. The effect of BSA on crop productivity was quantified by characterizing experimental conditions.

Results

In the published experiments, with biochar amendment rates generally <30 t ha^?1, BSA increased crop productivity by 11.0 % on average, while the responses varied with experimental conditions. Greater responses were found in pot experiments than in field, in acid than in neutral soils, in sandy textured than in loam and silt soils. Crop response in field experiments was greater for dry land crops (10.6 % on average) than for paddy rice (5.6 % on average). This result, associated with the higher response in acid and sandy textured soils, suggests both a liming and an aggregating/moistening effect of BSA.

Conclusions

The analysis suggests a promising role for BSA in improving crop productivity especially for dry land crops, and in acid, poor-structured soils though there was wide variation with soil, crop and biochar properties. Long-term field studies are needed to elucidate the persistence of BSA’s effect and the mechanisms for improving crop production in a wide range of agricultural conditions. At current prices and C-trading schemes, however, BSA would not be cost-effective unless persistent soil improvement and crop response can be demonstrated.     

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.