Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees, and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1,715 combined CDSs from whole‐genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain‐specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodeling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

BACKGROUND: Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. RESULTS: Total carotenoids progressively increased during fruit ripening up to ~55 μg g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. CONCLUSIONS: Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening

Dynamic protein trafficking to the cell wall

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed

Seeds of pomegranate, tomato and grapes: An underestimated source of natural bioactive molecules and antioxidants from agri-food by-products

Pomegranate, tomato and grape seeds are quantitatively relevant agri-food by-products rich in molecules beneficial to human health. To valorize this resource, the composition and antioxidant activity of seeds and deriving supercritical CO2 (SC-CO2) extracted oleoresins were evaluated. Grape seeds showed the highest content of total phenolic compounds (33.9 mg GAE/g), flavonoids (15.6 mg CE/g) and condensed tannins (14.0 mg CE/g), while tomato seeds presented the highest content of tocochromanols (159.6 μg/g). Grape seeds showed the highest total antioxidant activity (178.2 μmol TE/g), as evaluated by TEAC assay, followed by pomegranate (19.8 μmol TE/g) and tomato (9.8 μmol TE/g). Oleoresin yields obtained by SC-CO2 extraction from the seeds ranged between 3.1 (pomegranate) to 7.8 (tomato) g oleoresin/100 g. Total tocochromanols were abundant in pomegranate (2008 μg/g) and tomato (1769 μg/g) oleoresins; a relatively low amount was instead detected in the oleoresin extracted from grape seeds (636 μg/g). Carotenoids were not detected in all oleoresins. Pomegranate oleoresin had a higher antioxidant activity than the others. Mono- and polyunsaturated fatty acids were more abundant than saturated in all oleoresins, with the highest percentage of unsaturated fatty acids detected in pomegranate seed oleoresin (â\u88¼90%), mainly due to punicic acid (â\u88¼70%)

Expression of Exogenous GFP-CesA6 in Tobacco Enhances Cell Wall Biosynthesis and Biomass Production

Improved cellulose biosynthesis and plant biomass represent important economic targets for several biotechnological applications including bioenergy and biofuel production. The attempts to increase the biosynthesis of cellulose by overexpressing CesAs proteins, components of the cellulose synthase complex, has not always produced consistent results. Analyses of morphological and molecular data and of the chemical composition of cell walls showed that tobacco plants (F31 line), stably expressing the Arabidopsis CesA6 fused to GFP, exhibits a “giant” phenotype with no apparent other morphological aberrations. In the F31 line, all evaluated growth parameters, such as stem and root length, leaf size, and lignified secondary xylem, were significantly higher than in wt. Furthermore, F31 line exhibited increased flower and seed number, and an advance of about 20 days in the anthesis. In the leaves of F31 seedlings, the expression of primary CesAs (NtCesA1, NtCesA3, and NtCesA6) was enhanced, as well as of proteins involved in the biosynthesis of non-cellulosic polysaccharides (xyloglucans and galacturonans, NtXyl4, NtGal10), cell wall remodeling (NtExp11 and XTHs), and cell expansion (NtPIP1.1 and NtPIP2.7). While in leaves the expression level of all secondary cell wall CesAs (NtCesA4, NtCesA7, and NtCesA8) did not change significantly, both primary and secondary CesAs were differentially expressed in the stem. The amount of cellulose and matrix polysaccharides significantly increased in the F31 seedlings with no differences in pectin and hemicellulose glycosyl composition. Our results highlight the potentiality to overexpress primary CesAs in tobacco plants to enhance cellulose synthesis and biomass production