Debora Fontanini

Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Major proteinase hydrolysing gliadin during wheat germination

Abstract A proteinase, representing the bulk of the enzyme activity for the hydrolysis of gliadin, was extracted from endosperms isolated from germinated seeds (four days) and was purified by ion-exchange chromatography and preparative isoelectric focusing. The optimal pH for gliadin hydrolysis was 4.25. The M r , determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 30 000; the isoelectric point was 4.5. The enzyme activity was totally inhibited by E-64 and cystatin, while inhibitors of other classes of proteinases were barely effective or ineffective. The activity was stimulated by sulphhydryl compounds. The proteinase hydrolysed to small peptides the gliadins from durum and soft wheat seeds. Other protein substrates were weakly degraded or not degraded. The proteinase appears to belong to the cysteine class and to play a key role in the initial mobilization of the main reserve protein in the starchy endosperm.

Sequence determination of αs1-casein isoforms from donkey by mass spectrometric methods

Four co-eluting components, with experimentally measured M(r) of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkeys alpha(s1)-CNs and their sequences completely characterized, using the known mares alpha(s1)-CN (GenBank Acc. No. AAK83668; M(r) 23750.7 Da) as reference. The proteins with M(r) of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q(8)-->H and H(115)-->Y with respect to the mares alpha(s1)-CN. The other two components with M(r) 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu(33) and the Glu(34). The two alpha(s1)-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M(r) 24 406) and A(1) (201 amino acids; M(r) 24 278), whereas the two alpha(s1)-CNs without the pentapeptide were named variants B (197 amino acids; M(r) 23 786) and B(1) (196 amino acids; M(r) 23 658).

MS-based characterization of αs2-casein isoforms in donkey's milk†

The primary structure of four α(s2)-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkeys α(s2)-CN (GenBank Acc. No. CAV00691; M(r) 26,028 Da) as reference. Proteins, with experimentally measured M(r) of 25,429, 21,939, 25,203 and 21,713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, α-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.

Hydrolysis of Gliadin during Germination of Wheat Seeds

Summary The in vivo degradation of gliadin has been studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Both analyses show the appearance of a small number of degradation products and the early disappearance of some gliadin components during the course of germination. These components thus appear to be subject to preferential breakdown, the w-gliadin fraction being the first to be degraded. The in vitro degradation of total gliadin or selected gliadin fractions, performed by the main endopeptidase appearing during wheat germination, reveals a transient accumulation of intermediates, which were subsequently degraded to small peptides. These data suggest that the relatively small amount of intermediates formed during wheat germination might be due to the concerted action of the cysteine proteinase and of the carboxypeptidases. Since these enzymes have high activities during the course of seed germination, they might cause a rapid degradation of intermediates before they can accumulate.

A heterotetrameric alpha-amylase inhibitor from emmer (Triticum dicoccon Schrank) seeds

Plants have developed a constitutive defense system against pest attacks, which involves the expression of a set of inhibitors acting on heterologous amylases of different origins. Investigating the soluble protein complement of the hulled wheat emmer we have isolated and characterized a heterotetrameric α-amylase inhibitor (ETI). Based on mass spectrometry data, it is an assembly of proteins highly similar to the CM2/CM3/CM16 found in durum wheat. Our data indicate that these proteins can also inhibit exogenous α-amylases in binary assemblies. The calculated dissociation constants (K(i)) for the pancreatic porcine amylase- and human salivary amylase-ETI complexes are similar to those found in durum and soft wheat. Homology modeling of the CM subunits indicate structural similarities with other proteins belonging to the cereal family of trypsin/α-amylase inhibitors; a possible homology modeled structure for a tetrameric assembly of the subunits is proposed.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

Phytochemicals and antioxidant capacity in four Italian traditional maize (Zea mays L.) varieties

Abstract Flours of four pigmented (from orange to red and dark red) local Italian corns, studied for their soluble, soluble conjugate, and insoluble-bound phenols and flavonoids, showed a prevalence of the insoluble-bound fraction (70–80%). Correlations were found between the flours antioxidant capacity, measured with CUPRAC, FRAP, and DPPH methods, and soluble phenols and flavonoids content. A correlation was also found between ascorbic acid content and flours antioxidant power. Anthocyanins were present in small amounts in the red/dark red seeds; however, acid-alcohol assays and spectral analyses of pericarp extracts indicated the presence of red-brick phlobaphenes in these varieties. Spectrophotometrically quantified total carotenoids were significantly higher in one of the local varieties (Nano); RP-HPLC analyses indicated that the local varieties contained significantly higher amounts of zeaxanthin and β-carotene, and lower amounts of lutein, than a commercial line. Among local varieties, Nano expressed the highest levels of zeaxanthin, β-carotene, and β-cryptoxanthin.

Macro-grazer herbivory regulates seagrass response to pulse and press nutrient loading

Coastal ecosystems are exposed to multiple stressors. Predicting their outcomes is complicated by variations in their temporal regimes. Here, by means of a 16-month experiment, we investigated tolerance and resistance traits of Posidonia oceanica to herbivore damage under different regimes of nutrient loading. Chronic and pulse nutrient supply were combined with simulated fish herbivory, treated as a pulse stressor. At ambient nutrient levels, P. oceanica could cope with severe herbivory, likely through an increase in photosynthetic activity. Elevated nutrient levels, regardless of the temporal regime, negatively affected plant growth and increased leaf nutritional quality. This ultimately resulted in a reduction of plant biomass that was particularly severe under chronic fertilization. Our results suggest that both chronic and pulse nutrient loadings increase plant palatability to macro-grazers. Strategies for seagrass management should not be exclusively applied in areas exposed to chronic fertilization since even short-term nutrient pulses could alter seagrass meadows.