Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the α1-α2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. One of the proteins with an ESCRT-III like sequence pattern, which strikingly resembled Snf7 in its secondary structure, was studied more closely. We named the protein encoded by ORF YPL199c Etl1 (ESCRT-three-like 1). Etl1 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, Etl1 does not appear to be associated with lipid rafts, since it could be easily extracted from the membrane by Triton X-100 treatment. When ETL1 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. So far, despite a number of experiments, a bona fide ESCRT-III function could not be demonstrated for Etl1.

Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the α 1- α 2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. The protein encoded by ORF YPL199c strikingly resembled Snf7 in its secondary structure. Since this protein could be the ninth member of the ESCRT-III family in yeast, we called it Nbr9 (“number nine”). Nbr9 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, it is not associated with lipid rafts. When NBR9 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. However, despite a number of experiments, we do not have evidence at present that Nbr9 is part of an alternative ESCRT-III complex.### Competing Interest StatementThe authors have declared no competing interest.

Here, we examine the genetic interactions between ESCRT-III mutations in the yeast Saccharomyces cerevisiae. From the obtained interaction network, we make predictions about alternative ESCRT-III complexes. By the successful generation of an octuple deletion strain using the CRISPR/Cas9 technique, we demonstrate for the first time that ESCRT-III activity as a whole is not essential for the life of a yeast cell. Endosomal sorting complex required for transport (ESCRT)-III proteins are membrane remodeling factors involved in a multitude of cellular processes. There are eight proteins in yeast with an ESCRT-III domain. It is not clear whether the diverse ESCRT-III functions are fulfilled by a single ESCRT-III complex or by different complexes with distinct composition. Genetic interaction studies may provide a hint on the existence of alternative complexes. We performed a genetic mini-array screen by analyzing the growth phenotypes of all pairwise combinations of ESCRT-III deletion mutations under different stress conditions. Our analysis is in line with previous data pointing to a complex containing Did2/CHMP1 and Ist1/IST1. In addition, we provide evidence for the existence of a novel complex consisting of Did2/CHMP1 and Vps2/CHMP2. Some of the interactions on Congo red plates could be explained by effects of ESCRT-III mutations on Rim101 signaling.

The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our findings are in line with the current concepts about ESCRT-III, but some findings are unexpected and call for adjustments to the model. In particular, our data suggest that the distinction between bona fide ESCRT-III components and ESCRT-III associated proteins is not justified. We detected a single complex containing all ESCRT-III members (except of Chm7) with Did2 as its main component. The classical core components were present in equimolar amounts. Our analysis of the impact of single deletions on the composition of ESCRT-III confirmed the central role of Snf7 for ESCRT-III assembly. For the other ESCRT-III family members predictions could be made about their role in ESCRT-III assembly. Furthermore, our cell fractionation points to a role of Vps20 at the endoplasmic reticulum.

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.

Background and aims Only few data on the epicuticular waxes (EWs) of horsetails are available. This contribution therefore focuses on the wax micromorphology and chemical composition of Equisetum species of the subgenera Equisetum and Hippochaete.Methodology Distribution patterns and structural details of EW on the shoots were studied by scanning electron microscopy. After extraction with chloroform, the chemical composition of wax isolates was analysed by gas chromatography.Principal results Epicuticular wax crystals were non-oriented platelets or membraneous platelets. They were usually located on subsidiary cells of stomata and adjacent cells. Other parts of the shoots were covered mainly with a smooth wax film or small granules only. The chemical constituents found were alkanes, esters, aldehydes, primary alcohols and free fatty acids in a range of C-20-C-36 (in esters C-36-C-56). All species of the subgenus Hippochaete showed a similar pattern of fractions with high percentages of alkanes and aldehydes, whereas the subgenus Equisetum species had distinctly different wax compositions. Extracts from the internodes-surfaces without well-developed EW crystals and only few stomata-showed the lowest contents of aldehydes.Conclusions The covering with EW crystals will provide unhindered gas exchange and, combined with intracuticular wax, may prevent excess water loss during winter in the evergreen shoots of the subgenus Hippochaete. The results indicate that the Equisetum wax micromorphology and biosynthesis are comparable to EW of other pteridophyte classes and mosses.

As limited information is available about the relationship between microbial processes and community structure in tropical soils, we used 15N-DNA stable isotope probing (15N-DNA-SIP) to identify bacteria actively involved in decomposition of plant residues of different biochemical quality. 15N-labeled (90atom%) and unlabeled (control) maize (C-to-N ratio: 32; cellulose content: 24.9%) and soybean (15; 15.5%) leaf residues were incubated in a tropical Vertisol for 15days. Soil DNA was isolated, subjected to 15N-DNA-SIP and buoyant density-resolved DNA fractions were analyzed by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of selected DGGE bands. Residue addition induced new bands and changed relative intensity of already existing bands in 15N-enriched SIP fractions. Phylogenetic analysis of selected, cloned DGGE bands from ‘heaviest’ 15N-enriched fractions (57.8atom% (maize), 87.1atom% (soybean)) revealed that soils treated with maize residues were dominated by Pseudonocardia sp., while Arthrobacter sp. and Streptomyces sp. were found in the soybean residue treated soils. Sequences related to Bacillus sp. and Saccharopolyspora sp. were found in both organic residue treatments. Our study gave clear evidence that 15N-DNA-SIP combined with 16S rRNA gene-based community fingerprinting of density-resolved fractions and an unlabeled control was suited for detecting active bacteria involved in decomposition of complex maize and soybean residues. In conclusion, we could show that residue quality, inducing contrasting N assimilation by decomposing bacteria, was a substantial determinant of certain decomposing community members assayed in this study.

N-15-DNA stable isotope probing (N-15-DNA-SIP) combined with 18S rRNA gene-based community analysis was used to identify active fungi involved in decomposition of N-15-labeled maize and soybean litter in a tropical Vertisol. Phylogenetic analysis of N-15-labeled DNA subjected to 18S rRNA gene-based community fingerprinting showed that addition of maize residues promoted relatively slow-growing fungal decomposers (i.e. Penicillium spp., Aspergillus spp.), while addition of soybean residues promoted relatively fast-growing fungal decomposers (i.e. Fusarium spp., Mortierella spp.). Chaetomium spp. were dominant decomposers in both residue treatments. Therefore, we have clear evidence that specific members of the fungal community used N-15 derived from the two organic resources with contrasting biochemical quality for growth. Our study showed that N-15-DNA-SIP-based community analysis is a useful method to follow the fate of N from organic resources into the actively decomposing fungal community of soils. (C) 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.

Premise of the study : The succulent biome is highly fragmented throughout the Old and New World. The resulting disjunctions on global and regional scales have been explained by various hypotheses. To evaluate these, we used Thamnosma, which is restricted to the succulent biome and has trans-Atlantic and trans-African disjunctions. Its three main distribution centers are in southern North America, southern and eastern Africa including Socotra.Methods : We conducted parsimony, maximum likelihood, and Bayesian phylogenetic analyses based on chloroplast and nuclear sequence data. We applied molecular clock calculations using the programs BEAST and MULTIDIVTIME and biogeographic reconstructions using S-DIVA and Lagrange.Key results : Our data indicate a weakly supported paraphyly of the New World species with respect to a palaeotropical lineage, which is further subdivided into a southern African and a Horn of Africa group. The disjunctions in Thamnosma are mostly dated to the Miocene.Conclusions : We conclude that the Old-New World disjunction of Thamnosma is likely the result of long-distance dispersal. The Miocene closure of the arid corridor between southern and eastern Africa may have caused the split within the Old World lineage, thus making a vicariance explanation feasible. The colonization of Socotra is also due to long-distance dispersal. All recent Thamnosma species are part of the succulent biome, and the North American species may have been members of the arid Neogene Madro-Tertiary Geoflora. Phylogenetic niche conservatism, rare long-distance dispersal, and local differentiation account for the diversity among species of Thamnosma.

A field-scale manipulation experiment conducted for 16 years in a Norway spruce forest at Solling, Central Germany, was used to follow the long-term response of total soil bacteria, nitrate reducers and denitrifiers under conditions of reduced N deposition. N was experimentally removed from throughfall by a roof construction ('clean rain plot'). We used substrate-induced respiration (SIR) to characterize the active fraction of soil microbial biomass and potential nitrate reduction to quantify the activity of nitrate reducers. The abundance of total bacteria, nitrate reducers and denitrifiers in different soil layers was analysed by quantitative PCR of 16S rRNA gene, nitrate reduction and denitrification genes. Reduced N deposition temporarily affected the active fraction of the total microbial community (SIR) as well as nitrate reductase activity. However, the size of the total, nitrate reducer and denitrifier communities did not respond to reduced N deposition. Soil depth and sampling date had a greater influence on the density and activity of soil microorganisms than reduced deposition. An increase in the nosZ/16S rRNA gene and nosZ/nirK ratios with soil depth suggests that the proportion of denitrifiers capable of reducing N2O into N2 is larger in the mineral soil layer than in the organic layer.

Transport of litter carbon in the detritusphere might determine fungal abundance and diversity at the small scale. Rye residues were applied to the surface of soil cores with two different water contents and incubated at 10°C for 2 and 12 weeks. Fungal community structure was analysed by constructing clone libraries of 18S rDNA and subsequent sequencing. Litter addition induced fungal succession in the adjacent soil and decreased detectable fungal diversity mainly due to the huge supply of substrates. Ergosterol content and N-acetyl-glucosaminidase activity indicated fungal growth after 2 weeks. Simultaneously, the structure of the fungal community changed, with Mortierellaceae proliferating during the initial phase of litter decomposition. Ergosterol measurements were unable to detect this early fungal growth because Mortierellaceae do not produce ergosterol. In the late phase during decomposition of polymeric substrates, like cellulose and chitin, the fungal community was dominated by Trichocladium asperum. Water content influenced community composition only during the first 2 weeks due to its influence on transport processes in the detritusphere and on competition between fungal species. Our results underline the importance of species identification in understanding decomposition processes in soil.

Elevated dietary fructose intake, altered intestinal motility, and barrier function may be involved in the development of nonalcoholic fatty liver disease (NAFLD). Because intestinal motility and permeability are also regulated through the bioavailability of serotonin (5-HT), we assessed markers of hepatic injury in serotonin reuptake transporter knockout (SERT(-/-)) and wild-type mice chronically exposed to different monosaccharide solutions (30% glucose or fructose solution) or water for 8 wk. The significant increase in hepatic triglyceride, TNF-alpha, and 4-hydroxynonenal adduct as well as portal endotoxin levels found in fructose-fed mice was associated with a significant decrease of SERT and the tight-junction occludin in the duodenum. Similar effects were not found in mice fed glucose. In contrast, in SERT(-/-) mice fed glucose, portal endotoxin levels, concentration of occludin, and indices of hepatic damage were similar to those found in wild-type and SERT(-/-) mice fed fructose. In fructose-fed mice treated with a 5-HT3 receptor antagonist, hepatic steatosis was significantly attenuated. Our data suggest that a loss of intestinal SERT is a critical factor in fructose-induced impairment of intestinal barrier function and subsequently the development of steatosis.

Fungi play a major role in C and N cycling of plant residues, because of their ability to degrade complex substrates. Slow growing saprotrophic fungi are able to degrade lignin using extracellular enzymes and thereby gaining access to protected cellulose and hemi-cellulose compounds. On the other hand, fast growing opportunistic fungi (sugar fungi) are stimulated by easy accessible carbon sources. To evaluate the effect of plant residue quality on soil fungal community, an incubation experiment was conducted with highly 15N-enriched (99 atom%) plant residues of different quality, i.e. maize (C:N= 32, lignin:N=2.2) and soybean (C:N= 15, lignin:N=1.1), incorporated (1%) in a Vertisol soil, taken from a long-term field experiment carried out in Venezuela since 1997. The residues were incubated for 30 days (25 oC) at 40 % WHC. A control without residue was also used. The ergosterol content was measured after 3, 7, 15 and 30 days. DNA was extracted and the active and passive fungal community composition is being analysed by using the 15N-DNA stable isotopic probing (SIP) and molecular (DGGE, cloning and sequencing) techniques. Residues additions stimulated soil fungal activities and the quality of residues influenced the microbial biomass. The fungal biomass was higher in the soybean treatment already at early stages of decomposition compared to the maize treatment. This seems to indicate that quality of residues affected the fungal community, i.e. in soybean it is probably composed mainly of sugar fungi and in maize probably dominated by slow growing lignocellulytic fungi. Further DNA analysis should help clarifying this difference. High correlation was found between fungal biomass and enzymes activities involved in C-cycling suggesting that fungi play a major role in the recycling of C and nutrients in the tropical soil.

Epicuticular wax in sugar cane leaves is present as platelets, short furrowed rodlets and massive prominent crystalloids which consist of longitudinally aggregated rodlets. In rice, the leaf surfaces are uniformly covered by platelets in a dense, irregular arrangement. The wax crystalloids of sugar cane are affected by CHCl3 extraction at room temp. only to a little extend and less than one tenth of total epicuticular wax is obtained by this treatment. Significant disturbance of the wax structure takes place in rice, the degree of disintegration and loss of crystalloids differs between cultivars. Compared to sugar cane, the relative amounts of waxes extracted are considerably greater. Primary alcohols and esters are the compound classes of long chain wax constituents preferably extracted by CHCl3 at room temp. Complete removal of all epicuticular waxes is achieved by extraction with CHCl3 at 60degreesC in rice and sugar cane as well. Whilst the amounts of aldehydes are small in the first extracts, they are the main constituent class obtained by extraction with hot CHCl3. This suggests that aldehydes are responsible for the incomplete extraction with CHCl3 at room temp. because of their presence in an insoluble, i.e. polymeric form. They dissolve at the elevated temperature by cleavage of polymer bonds. This is in agreement with previous results on the Solubility of polymeric aldehydes in epicuticular wax from the culms of sugar cane. The relative solvent resistance of wax crystalloids reinforced by polymeric aldehydes may be indicative of improved stability and corrosion resistance under natural conditions, too. This could be of importance concerning the effects of stable and well developed wax crystalloids on leaf surfaces which include water repellency and therefore unhindered gas exchange as Well as reduced risk of contamination and microbial infections.