The C-terminal extension of the beta 7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. beta 7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10 Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4Cterminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.info:eu-repo/semantics/publishedVersio

PACemakers of proteasome core particle assembly

The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, P subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta 7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta 7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.Fundacao para a Ciencia e Tecnologia; Deutsche Forschungsgemeinschaf

Hsp70 and Hsp110 chaperones promote early steps of proteasome assembly

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete alpha-ring that functions as a template for beta subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of alpha subunits in S. cerevisiae. Instead, we found that the CP subunits alpha 1, alpha 2, and alpha 4 each form independent small complexes. Purification of such complexes containing alpha 4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing alpha 1, alpha 2, and alpha 4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (alpha 3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with alpha subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual alpha subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.info:eu-repo/semantics/publishedVersio

Populational analysis of Saccharomyces cerevisiae strains from different appellations of origin and grape varieties by microsatellite analysis.

The objective of the present study was to evaluate populational relationships among Saccharomyces cerevisiae strains isolated from some of the Portuguese most important grapevine varieties in different appellations of origin, using polymorphic microsatellites. 
One hundred ninety two grape samples were collected during the 2006 and 2007 harvest season in the Vinho Verde (grape varieties: Arinto, Alvarinho, Avesso, Loureiro, Touriga Nacional) Bairrada (grape varieties: Arinto, Baga, Castelão Francês, Maria Gomes, Touriga Nacional) Alentejo (grape varieties, Aragonês, Trincadeira, Touriga Nacional), Terras do Sado (grape variety Castelão) Bucelas (grape variety Arinto) and Estremadura (grape varieties: Arinto, Aragonês, Castelão, Trincadeira, Touriga Nacional) appellations of origin. From the final stage of spontaneous fermentations, 2820 yeast isolates were obtained, mainly belonging to the species S. cerevisiae. An initial genetic screen, based on mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and/or interdelta sequence analysis was followed by microsatellite analysis of strains with unique genetic profiles, using 10 highly polymorphic microsatellites. Our results showed that microsatellite analysis revealed a high resolution populational screen, showing that genetic differences and populational structures among S. cerevisiae populations derived from both “diagnostic” vineyard-, specific alleles and the accumulation of small allele-frequency differences across ten microsatellite loci. Heterozygosity was three to four times lower than the expected value, confirming the strong populational substructuring. The presented large-scale approach shows that each vineyard contains differentiated S. cerevisiae populations, showing the occurrence of specific native strains that can be associated with a terroir. 

Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762).
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Effects of soil contamination by trace elements on white poplar progeny: seed germination and seedling vigour

11 páginas.-- 2 figuras.-- 2 tablas.-- 62 referencias.-- Electronic supplementary material in the online version of this article (doi:10.​1007/​s10661-015-4893-8) contains supplementary material, which is available to authorized users.Seed germination is considered a critical phase in plant development and relatively sensitive to heavy metals. White poplar (Populus alba) trees tend to accumulate Cd and Zn in their tissues. We tested if soil contamination can affect P. alba progeny, reduced seed germination and explored the distribution of mineral elements in the seed. For this purpose, fruits and seeds from female P. alba trees were selected from two contaminated and one non-contaminated areas. Seeds from all the sites were germinated using only water or a nutritive solution (in vitro). Concentrations of nutrients and trace elements in the fruits and seeds were analysed. Seedling growth in vitro was also analysed. Finally, a mapping of different elements within the poplar seed was obtained by particle-induced X-ray emission (PIXE). Germination was similar between different progenies, refuting our hypothesis that seeds from a contaminated origin would have reduced germination capacity compared to those from a non-contaminated site. Seedling growth was not affected by the contaminated origin. Cadmium and Zn concentrations in fruits produced by P. alba trees in the contaminated sites were higher than by those from the non-contaminated site. However, the nutritional status of the trees was adequate in both cases. Cd in seedlings was higher in those from contaminated soils although lower than in fruits, indicating a certain exclusion from seeds. Preliminary results of the PIXE technique showed that Al and Zn were distributed uniformly in the seeds (Cd was not detected with this technique), while the nutrients P and S were concentrated in the cotyledons.We thank JM Alegre for helping in the field work and Patricia Puente for helping in the lab work.We thank the National Accelerator Centre (CNA) of Seville for the analytical facilities. This study was supported by CGL2011-30285-C02 project, funded by the CICYT of the Spanish Ministerio de Ciencia e Innovación and European FEDER funds.Peer reviewe

2,2′,5,5′-Tetra­methyl-1,1′-(hexane-1,6-di­yl)di-1H-pyrrole

The mol­ecule of the title compound, C18H28N2, composed of two 2,5-dimethyl­pyrrole groups linked by a hexane chain, lies across a crystallographic inversion centre. The mean plane of the pyrrole ring is almost perpendicular to the mean plane of the central chain, making a dihedral angle of 89.09 (8)°. The crystal structure is stabilized by inter­molecular C—H⋯π inter­actions

Performance of Quercus suber L. at nursery stage - application of two bio-inoculants under two distinct environments

Key message - Despite the fact that the technique of application of bioinoculants improved the quality of Quercus suber L. seedlings produced in nurseries, these benefits are dependent on the ecological conditions of the site and the composition of the applied inoculum, which interferes with the profile of the local fungal community. Context - Quercus suber L. plays a key ecological and socio-economical role in the Iberian Peninsula. Symbiotic ectomycorrhizal fungi-ECM are crucial partners of several tree species, and assessing the efficacy of bioinoculants at nursery stage helps devising tools to increase plant resilience. Aims - The aim of this study was to compare the effects of two inocula formulations of mixed ECM fungi and bacteria on the quality of seedlings produced in two forest nurseries, differing in environmental conditions and forest embedment. Methods - Quercus suber L. seedlings were inoculated with a commercial product containing Pisolithus tinctorius (Pers) Coker Couch - Scleroderma sp., and six bacterial species and with a non-commercial fungal and bacterial dual inoculum (Suillus granulatus (L.) Roussel + Mesorhizobium sp.). Biometric and nutritional parameters and morphological quality indexes were determined on seedlings. The ECMcommunity was assessed by denaturing gradient gel electrophoresis and cloning-sequencing. Results - In both nurseries, the seedling quality index in inoculated was up to 2-fold higher than in non-inoculated seedlings. Plant biomass differed significantly among nurseries. The inoculum influenced the profile of the fungal community. S. granulatus and P. tinctorius persisted for 6 months in the inoculated seedlings. Conclusion- The nursery ecosystem influenced plant growth. Inoculation treatments increased plant performance; however, the dual inoculum resulted in more consistent improvements of Q. suber at nursery stage, highlighting the importance of inocula selection.info:eu-repo/semantics/publishedVersio

Two genotypes of mycorrhizal Pinus pinaster respond differently to cadmium contamination

Fertilization is one of the main anthropogenic sources of Cd accumulation in agricultural soils and when toxic levels have been reached, food crop production is no longer viable. Adequate strategies for the forestation of agricultural metal contaminated sites are of vital importance. The aim of this work was to evaluate the response of two different genotypes of P. pinaster (A and B) to Cd contamination and to assess how inoculation with ectomycorrhizal fungi influenced each genotype. Seedlings were exposed to soil contaminated at different levels of Cd. At 30 mg Cd kg-1 non-inoculated genotype A accumulated more Cd in the shoots. At the lowest Cd concentration S. bovinus decreased Cd shoot concentration and increased aboveground development in both genotypes. At the highest Cd dosage inoculation with R. roseolus decreased Cd concentration in the roots of genotype B whereas the opposite occurred in genotype A. The results from this study suggest that the selection of an adequate combination between genotype and associated mycobionts may be an important biotechnological tool to enhance the efficiency of forestation and phytoremediation processes of degraded land using P. pinaster

Inoculation with ectomycorrhizal fungi affects Pinus pinaster performance under cadmium exposure

Afforestation of contaminated sites can represent a valuable approach to restore degraded ecosystems. Studies on the response of woody species to heavy metal contamination in soil are scarce compared to crop species. Cadmium is one of the most toxic heavy metals and its hazardous effects are well known. The aim of this work was to evaluate Pinus pinaster performance on Cd contaminated soil (15 and 30 mg Cd kg-1) and determine whether inoculation with two ectomycorrhizal fungi, Suillus bovinus and Rhizopogon roseolus influenced such response. Regarding non-inoculated seedlings, Cd exposure led to a lower shoot biomass and metal accumulation on the root system was proportional to its concentration in the soil. Inoculation with S. bovinus was the most favorable treatment for P. pinaster development by enhancing shoot development up to 1.3-fold in contaminated soil. Inoculation with R. roseolus increased Cd concentration in the shoots with no significant effect in any of the biometric traits studied. Metal accumulation on the shoots and roots of P. pinaster seedlings was significantly affected by the interaction between mycorrhizal inoculation and the Cd concentration to which the seedlings were exposed. Results from this study show that inoculation with selected ECM fungi can influence the performance of P. pinaster under Cd exposure and that this biotechnological tool could be of great value for plant establishment in contaminated sites

Simultaneous partial nitrification and 2-fluorophenol biodegradation with aerobic granular biomass: reactor performance and microbial communities

Supplementary data associated with this article can be found, in the online version, at: http://dx.doi.org/10.1016/j.biortech.2017.03.173.An aerobic granular bioreactor was operated for over 4 months, treating a synthetic wastewater with a high ammonium content (100 mg L-1). The inoculum was collected from a bioreactor performing simultaneous partial nitrification and aromatic compounds biodegradation. From day-56 onwards, 2-fluorophenol (2-FP) (12.4 mg L-1) was added to the feeding wastewater and the system was bioaugmented with a 2-FP degrading bacteria (Rhodococcus sp. FP1). By the end of operation, complete 2-FP biodegradation and partial nitrification were simultaneously achieved. Aerobic granules remained stable over time. During the 2-FP loading, a shift in the community structure occurred, coinciding with the improvement of 2-FP degradation. DGGE analysis did not allow to infer on the bioaugmented strain presence but pyrosequencing analysis detected Rhodococcus genus by the end of operation. Together with other potential phenolic-degraders within granules, these microorganisms were probably responsible for 2-FP degradation.This work was supported by Portuguese Funds from FCT - Fundação para a Ciência e a Tecnologia through the strategic funding of UID/Multi/ 50016/2013 and UID/BIO/04469/2013 units and COMPETE 2020 (POCI-01-0145-FEDER-006684) and Spanish Funds from Ministerio de Educación y Ciencia through the ONLYBIO project (CTQ2011-24745/PPQ). C.L. Amorim and D.P Mesquita wish to acknowledge the research grant from FCT (Ref. SFRH/BPD/96481/2013 and SFRH/BPD/82558/2011, respectively). C. Ramos wants to thank the Universitat Autònoma de Barcelona for his internship grant. The authors also thank to COST Action ES1202: Conceiving Wastewater Treatment in 2020 - Energetic, environmental and economic challenges; BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte; and to TRITON thematic network (316RT0508) from the CYTED Programme. Thanks to Dr R.C. Pullar for helping to edit the English.info:eu-repo/semantics/publishedVersio