The C-Domain of Oleuropein ß-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus.

Oleuropein, a terpene-derived glycosylated secoiridoid biosynthesized exclusively by members of the Oleaceae family, is involved in a two-component defense system comprising a ß-glucosidase that activates oleuropein into a toxic glutaraldehyde-like structure. Oleuropein and its deglycosylated derivatives have high pharmaceutical interest. In this study we determined that the in planta heterologous expressed OeGLU, an oleuropein-specific ß-glucosidase from olive (Olea europaea), had enzymatic kinetics similar to the olive native enzyme. The C terminus encompassing the nuclear localization signal sequesters the enzyme in the nucleus, and predetermines the protein-protein recognition and homodimerization. Biochemical analysis revealed that OeGLU is a homomultimer with high Mr In silico prediction modeling of the complex structure and bimolecular fluorescence complementation analyses revealed that the C terminus of OeGLU is essential for the proper assembly of an octameric form, a key conformational feature that determines the activity of the enzyme. Our results demonstrate that intrinsic characteristics of the OeGLU ensure separation from oleuropein and keep the dual-partner defensive system conditionally inactive. Upon cell destruction, the dual-partner defense system is activated and olive massively releases the arsenal of defense.

A defence-related Olea europaea ß-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent.

Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a ß-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein ß-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a ß-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (ß/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the ß-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products.

Comparative transcriptomic analysis reveals conserved programmes underpinning organogenesis and reproduction in land plants.

The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.

Insights into the role of quorum sensing in food spoilage.

Food spoilage is a consequence of the degrading enzymatic activity of some food-associated bacteria. Several proteolytic, lipolytic, chitinolytic, and pectinolytic activities associated with the deterioration of goods are regulated by quorum sensing, suggesting a potential role of such cell-to-cell communication in food spoilage. Here we review quorum sensing signaling molecules and methods of their detection and quantification, and we provide insights into the role of quorum sensing in food spoilage and address potential quorum sensing inhibitors that might be used as biopreservatives.

The use of indigenous Saccharomyces cerevisiae and Starmerella bacillaris strains as a tool to create chemical complexity in local wines.

The performance of two vineyard strains, Saccharomyces cerevisiae SacPK7 and Starmerella bacillaris StbPK9, was evaluated in laboratory and pilot scale fermentations of Cretan grape must under the following inoculation schemes: single inoculation of SacPK7 (IS), simultaneous inoculation of StbPK9 and SacPK7 (SM), and sequential inoculation of StbPK9 followed by SacPK7 (SQ). Un-inoculated (spontaneous) fermentations (SP) and fermentations inoculated with control S. cerevisiae strains (CS) were also conducted as reference. Star. bacillaris not only did not restrict but also slightly promoted the growth of S. cerevisiae when the two strains were co-inoculated at equal quantities. On the contrary, the SQ inoculation scheme conferred a competitive advantage to Star. bacillaris over S. cerevisiae, which maximum population was reduced, while increased levels of Star. bacillaris were recorded. The fermentation kinetics were also affected, accordingly. The completion of fermentation was faster in SM, IS and CS ferments than in SQ and SP. Ethanol accumulation had a predominant role in the early death of Star. bacillaris, since its growth was similarly arrested irrespective of the dominating yeast species, the magnitude of yeast population or the availability of energy sources. Interestingly, the inoculation scheme applied significantly affected the chemical profiles of the resulting wines. SQ produced the most divergent chemical profile in sterile must, with glycerol, acetic acid, acetaldehyde, residual glucose, malic acid, ethyl acetate and higher alcohols being the key compounds affected by the prolonged activity of StbPK9. In pilot scale ferments, the indigenous S. cerevisiae produced twice as high levels of esters and higher alcohols compared to the commercial starter. Star. bacillaris further increased the levels of ethyl esters in the respective ferments. The use of a mixed S. cerevisiae/Star. bacillaris starter culture instead of S. cerevisiae alone enhanced the chemical complexity of Cretan local wine. The magnitude of differentiation was even higher when the addition of Star. bacillaris preceded that of S. cerevisiae. The highest divergence in analytical profiles was recorded between wines produced by native strain combinations and commercial S. cerevisiae. Present results show that the use of indigenous yeast formulations provides significant diversification to local wines, in line with the microbial terroir concept and recent observations that indigenous yeast strains may confer regional characters to wines.

Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis.

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.

Flow cytometry as a diagnostic tool in the early diagnosis of aggressive lymphomas mimicking life-threatening infection.

Aggressive lymphomas can present with symptoms mimicking life-threatening infection. Flow cytometry (FC) is usually recommended for the classification and staging of lymphomas in patients with organomegaly and atypical cells in effusions and blood, after the exclusion of other possible diagnoses. FC may also have a place in the initial diagnostic investigation of aggressive lymphoma. Three cases are presented here of highly aggressive lymphomas in young adults, which presented with the clinical picture of fever of unknown origin (FUO) in patients severely ill. All followed a life-threatening clinical course, and two developed the hemophagocytic syndrome (HPS), but microbiological, immunological, and morphological evaluation and immunohistochemistry (IHC) failed to substantiate an early diagnosis. FC was the technique that provided conclusive diagnostic evidence of lymphoma, subsequently verified by IHC. Our experience with these three cases highlights the potential role of FC as an adjunct methodology in the initial assessment of possible highly aggressive lymphoma presenting with the signs and symptoms of life-threatening infection, although the definitive diagnosis should be established by biopsy. In such cases, FC can contribute to the diagnosis of lymphoma, independently of the presence of HPS.