Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.     

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca2+ homeostasis in human erythroleukemia cells

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca²⁺ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca²⁺ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca²⁺-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin.Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca²⁺.     

Wet-dry cycling extends seed persistence by re-instating antioxidant capacity

Seeds in the field experience wet-dry cycling that is akin to the well-studied commercial process of seed priming in which seeds are hydrated and then re-dried to standardise their germination characteristics. To investigate whether the persistence (defined as in situ longevity) and antioxidant capacity of seeds are influenced by wet-dry cycling, seeds of the global agronomic weed Avena sterilis ssp. ludoviciana were subjected to (1) controlled ageing at 60% relative humidity and 53.5°C for 31 days, (2) controlled ageing then priming, or (3) ageing in the field in three soils for 21 months. Changes in seed viability (total germination), mean germination time, seedling vigour (mean seedling length), and the concentrations of the glutathione (GSH) / glutathione disulphide (GSSG) redox couple were recorded over time. As controlled-aged seeds lost viability, GSH levels declined and the relative proportion of GSSG contributing to total glutathione increased, indicative of a failing antioxidant capacity. Subjecting seeds that were aged under controlled conditions to a wet-dry cycle (to ?1 MPa) prevented viability loss and increased GSH levels. Field-aged seeds that underwent numerous wet-dry cycles due to natural rainfall maintained high viability and high GSH levels. Thus wet-dry cycles in the field may enhance seed longevity and persistence coincident with re-synthesis of protective compounds such as GSH.     

Analysis of a range of catabolic mutants provides evidence that phytanoyl-coenzyme A does not act as a substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex in Arabidopsis during dark-induced senescence

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex.     

A new freshwater mite of the marine genus Halacarellus (Acari: Halacaridae) from the Austrian Alps (Styria, Gesäuse National Park): Description and reflections on its origin

A new halacarid species, Halacarellus fontinalis n. sp., from a spring in the Gesäuse National Park, Austria is described. The species is characterized by three pairs of large, equal-sized acetabula and slender claws and is expected to have evolved in the Tertiary from a Tethyan-Paratethyan Halacarellus species. CaspihalacarusViets, 1928, with a single species, C. hyrcanusViets, 1928, has large and external acetabula similar to those in H. fontinalis. Caspihalacarus is synonymized with Halacarellus.     

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.     

Trafficking of the myrosinase‐associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL‐like lipase implicated in glucosinolate metabolism through its association with the β‐glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild‐type plants suggested that GLL23 is normally post‐translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase‐associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi‐localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER–Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.     

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.