Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.Lignin is a major structural and protective component of plant cell walls. Lignin exists as a polymer of mainly three hydroxycinnamyl alcohols and related compounds, referred to as monolignols. The most common monolignols are coniferyl, sinapyl, and p-coumaryl alcohol (Ralph et al., 2004; Vanholme et al., 2010). After polymerization, structures derived from those compounds are referred to as guaiacyl, syringyl, and p-hydroxyphenyl subunits, respectively. The specific composition of lignin subunits varies among species, tissues, and developmental stages. Gymnosperm trees produce lignin that is primarily made of guaiacyl subunits, angiosperm trees contain guaiacyl and syringyl subunits, whereas grasses contain guaiacyl and syringyl subunits with small amounts (approximately 5%) of p-hydroxyphenyl residues. This observed variation in subunit composition across species may reflect the heterogeneity in substrate specificity and kinetic parameters among various monolignol biosynthetic enzymes (Weng et al., 2008).Biosynthesis of the monolignols occurs via the phenylpropanoid pathway using Phe precursors (Vanholme et al., 2010). Phe ammonia lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from Phe (Vanholme et al., 2010). Grasses can bypass cinnamate-4-hydroxylase by using Tyr as a substrate for Phe ammonia lyase (Neish, 1961; Rösler et al., 1997). The hydroxycinnamoyltransferase (HCT) enzymes exchange the CoA functionality esterified to p-coumaric acid with shikimic or quinic acid to allow for the subsequent conversion of the p-coumaroyl moiety to a caffeoyl moiety by p-coumarate-3′-hydroxylase (C3′H). The hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferases (HSTs) exhibit preference for shikimate, whereas the hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferases prefer quinate as a substrate (Sander and Petersen, 2011). Subsequent reactions ultimately lead to coniferyl and sinapyl alcohol via reduction of the γ-carbon on the propane side chain and substitution of the C3 and C5 positions of the phenol ring (Boerjan et al., 2003).Sorghum (Sorghum bicolor) is an attractive bioenergy crop with typical dry biomass yields between 20 and 25 Mg ha⁻¹ and yields as high as 40 Mg ha⁻¹ possible under optimal conditions (Venuto and Kindiger, 2008). Moreover, sorghum utilizes nitrogen-based fertilizer more efficiently than maize (Zea mays) and sugarcane (Saccharum officinarum), leading to less groundwater contamination and lower CO₂ emission (Propheter and Staggenborg, 2010; Wortmann and Regassa, 2011). Overall, sorghum has a higher sugar yield potential per land area and requires less water for growth than maize, allowing it to grow in a more diverse range of environments (Saballos, 2008). The sorghum genome sequence has been released (Paterson et al., 2009), and Targeting Induced Local Lesions in Genomes populations exist (Xin et al., 2008) in which various cell wall mutants have been identified (Sattler et al., 2012; Vermerris and Saballos, 2012).A detailed understanding of the catalytic mechanism of phenylpropanoid-related enzymes will enable the targeted modification of lignin subunit composition. The presence of lignin poses a major obstacle to the production of biofuels and chemicals from lignocellulosic biomass, because of its ability to hinder the activity of enzymes required to degrade cellulose to sugars that can be fermented for ethanol production (Yang and Wyman, 2004; Berlin et al., 2006). Genetic modification of plant cell wall composition, especially lignin content and subunit composition, has been shown to improve biomass conversion to fermentable sugars (Chen and Dixon, 2007; Vermerris et al., 2007; Jung et al., 2012). In particular, HCT silencing in Arabidopsis (Arabidopsis thaliana) causes an accumulation of p-hydroxyphenyl residues in the lignin and decreased content of guaiacyl and syringyl residues, leading to a dwarf phenotype (Li et al., 2010). Down-regulation of HCT has also been shown to result in decreased plant growth in alfalfa (Medicago sativa; Shadle et al., 2007). Concomitantly, ruminant digestibility and the yield of fermentable sugars following enzymatic saccharification increased (Chen and Dixon, 2007; Shadle et al., 2007). Reduced HCT activity may alter cell wall polymer interactions and allow better access of cellulolytic enzymes to the cellulose. Therefore, it has the potential to reduce the energy and processing costs associated with the conversion of biomass to fuels and chemicals. However fine-tuning will be necessary to limit the negative impacts on plant growth, which will require a detailed understanding of the catalytic mechanism of HCT.Given the difference in lignin subunit composition among different species and the prominence of grasses among dedicated bioenergy crops, we have focused on elucidating the crystal structure and activity of monolignol-related enzymes of sorghum, starting with the HST-like HCT. HCT belongs to the BAHD superfamily of plant-specific acyl-CoA-dependent acyltransferases (Ma et al., 2005; D’Auria, 2006). However, the BAHD superfamily has functionally and structurally diverse members that frequently possess little (as low as 10%) sequence identity among them (St-Pierre and Luca, 2000). Recent studies led to the crystal structure of the HST-like HCT from robusta coffee (Coffea canephora), an angiosperm dicot with a binding pocket elucidated by molecular docking and mutagenesis (Lallemand et al., 2012). In this report, we present the three-dimensional structures of HCT in its apo-form and ternary complex, supplemented by mutagenic studies to elucidate its reaction mechanism and structural relationship to other members in this growing functional class.     

First Record of Bourgelatia diducta (Nematoda: Chabertiidae) from Wild Boars in the Republic of Korea

This study describes the first record of Bourgelatia diducta (Nematoda: Chabertiidae) from wild boars in the Republic of Korea (=South Korea). Gastrointestinal tracts of 87 Korean wild boars (Sus scrofa coreanus) hunted in mountains in the south-western part of South Korea between 2009 and 2012 were examined for their visceral helminths. B. diducta, as identified by morphological characteristics of the head and tail, were recovered from the large intestine of 47 (54%) wild boars. The average length of adult female worms was 11.3±0.87 mm and the thickest part of the body measured 0.54±0.04 mm in maximum width, while those of males were 9.8±0.72 and 0.45±0.03 mm, respectively. The characteristic J-shaped type II ovejector was observed in females, and the type II dorsal ray with 2 rami on each side of the median fissure was uniquely seen in males. The buccal capsule was small, relatively thin-walled, cylindrical, very short, and ring-shaped. The externodorsal ray arose from a common stem with the dorsal ray. The cervical groove was absent. The anterior extremity was equipped with 20-22 external corona radiata, 4 cephalic papillae and 2 lateral amphids around the mouth. The eggs were 66.0×38.9 µm in average size. By the present study, B. diducta (Nematoda: Chabertiidae) is recorded for the first time in South Korea. Additionally, morphological characteristics and identification keys provided in the present study will be helpful in the faunistic or taxonomic studies for strongylid nematodes related.     

Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01～1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with ZnO NPs (0.01～1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.     

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.     

Effects of actinobacteria on plant disease suppression and growth promotion

Biological control and plant growth promotion by plant beneficial microbes has been viewed as an alternative to the use of chemical pesticides and fertilizers. Bacteria and fungi that are naturally associated with plants and have a beneficial effect on plant growth by the alleviation of biotic and abiotic stresses were isolated and developed into biocontrol (BCA) and plant growth-promoting agents (PGPA). Actinobacteria are a group of important plant-associated spore-forming bacteria, which have been studied for their biocontrol, plant growth promotion, and interaction with plants. This review summarizes the effects of actinobacteria as BCA, PGPA, and its beneficial associations with plants.     

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.     

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington’s disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.