Characterization of olive seed storage proteins

At present little is known about olive seed storage proteins (SSPs). A better understanding of olive SSPs will be important for future biotechnology efforts. In the present study, we first developed a protocol relied on chloroform for preparing protein samples free of lipids from lipid-rich olive seeds. Then, we characterized olive SSPs by SDS-PAGE, N-terminal sequencing and immunoblot. Two smaller subunits (20 and 21.5 kD) of SSPs were purified to homogeneity and used for antibody production or N-terminal sequencing. N-terminal sequencing confirmed that major olive SSPs are 11S globulins. Moreover, the components and size distribution of SSPs are identical among several olive cultivars examined, suggesting that their synthesis is highly conserved in this species. Olive SSPs are soluble in aqueous alcohol, with limited solubility in water and dilute salt. Thus, despite their homology with globulins, olive SSPs are similar in solubility to prolamins and different from globulins in other dicot plants. Finally, the accumulation of olive SSPs during fruit maturation was examined. Our results revealed that the accumulation of SSPs is time-dependent and tissue-specific, and only 105 days after pollination (DAP), did individual components of SSPs synthesize substantially, and accumulate rapidly in large quantities over a short period of time. Our results suggest that a 36 kD protein is the precursor of olive SSPs, and 90–105 DAP seems to be a crucial transition period (from a precursor to mature subunits) for the accumulation of SSPs.     

抗、感绿豆象绿豆种子差异蛋白的分离与鉴定

为寻找抗绿豆象绿豆种子中的重要抗虫成分,以抗虫绿豆晋绿7号、B20及感虫绿豆潍绿2117为试验材料,采用蛋白质双向电泳(2-DE)和质谱技术,对抗、感虫绿豆中差异蛋白及功能进行了鉴定与分析。结果表明,抗、感虫绿豆中差异蛋白表达量超过2.5倍的点共有15个。其中6个蛋白点通过数据库得到了成功鉴定,涉及3种蛋白质,分别为8S球蛋白(α亚型和β亚型)、核酮糖1,5-二磷酸羧化酶/加氧酶(RuBis CO)亚基结合蛋白和合成淀粉酶与胰蛋白酶两种抑制剂的前体多肽链。蛋白点B49(即8S球蛋白α亚型)和B31(RuBis CO亚基结合蛋白)在抗、感虫绿豆中的差异表达量分别达到10 000倍和23倍。抗虫绿豆中8S球蛋白α亚型及β亚型、RuBis CO伴侣蛋白及胰蛋白酶抑制剂的前体作为抗虫物质影响了绿豆象的生长发育甚至导致其死亡,与抗虫性的量效关系及联合效应还需进一步验证。     

Fruit and seed heteromorphism in the cold desert annual ephemeral Diptychocarpus strictus (Brassicaceae) and possible adaptive significance

Background and Aims

Diptychocarpus strictus is an annual ephemeral in the cold desert of northwest China that produces heteromorphic fruits and seeds. The primary aims of this study were to characterize the morphology and anatomy of fruits and seeds of this species and compare the role of fruit and seed hetermorphism in dispersal and germination.

Methods

Shape, size, mass and dispersal of siliques and seeds and the thickness of the mucilage layer on seeds were measured, and the anatomy of siliques and seeds, the role of seed mucilage in water absorption/dehydration, germination and adherence of seeds to soil particles, the role of pericarp of lower siliques in seed dormancy and seed after-ripening and germination phenology were studied using standard procedures.

Key Results

Plants produce dehiscent upper siliques with a thin pericarp containing seeds with large wings and a thick mucilage layer and indehiscent lower siliques with a thick pericarp containing nearly wingless seeds with a thin mucilage layer. The dispersal ability of seeds from the upper siliques was much greater than that of intact lower siliques. Mucilage increased the amount of water absorbed by seeds and decreased the rate of dehydration. Seeds with a thick mucilage layer adhered to soil particles much better than those with a thin mucilage layer or those from which mucilage had been removed. Fresh seeds were physiologically dormant and after-ripened during summer. Non-dormant seeds germinated to high percentages in light and in darkness. Germination of seeds from upper siliques is delayed until spring primarily by drought in summer and autumn, whereas the thick, indehiscent pericarp prevents germination for >1 year of seeds retained in lower siliques.

Conclusions

The life cycle of D. strictus is morphologically and physiologically adapted to the cold desert environment in time and space via a combination of characters associated with fruit and seed heteromorphism.     

A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in developing seeds of Arabidopsis thaliana and oil crops. Compartmentation of primary metabolism in developing embryos poses a significant challenge for testing this hypothesis and for the engineering of seed biomass production. It also raises the question whether there is a preferred route of carbon from imported photosynthate to seed oil in the embryo. Plastidic pyruvate kinase catalyzes a highly regulated, ATP-producing reaction of glycolysis. The Arabidopsis genome encodes 14 putative isoforms of pyruvate kinases. Three genes encode subunits alpha, beta(1), and beta(2) of plastidic pyruvate kinase. The plastid enzyme prevalent in developing seeds likely has a subunit composition of 4alpha4beta(1), is most active at pH 8.0, and is inhibited by Glu. Disruption of the gene encoding the beta(1) subunit causes a reduction in plastidic pyruvate kinase activity and 60% reduction in seed oil content. The seed oil phenotype is fully restored by expression of the beta(1) subunit-encoding cDNA and partially by the beta(2) subunit-encoding cDNA. Therefore, the identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into oil, suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty acids.     

Induction of vivipary in Arabidopsis by silique culture: implications for seed dormancy and germination

Culture of excised fruits (siliques) of different ages of Arabidopsis thaliana in a solidified mineral salt medium supplemented with vitamins, myo-inositol, and 3% sucrose induces vivipary. Whereas early stage and immature embryos complete their full development before germinating viviparously in seeds enclosed in the silique, mature green embryos enclosed in green ovules germinate without further growth in culture. Vivipary is not observed in cultured siliques enclosing brown ovules with yellowish mature embryos inside. Suggestive of a role for abscisic acid in preventing vivipary on the mother plant, addition of the hormone to the culture medium is found to inhibit vivipary in cultured siliques. Although dried green ovules enclosing mature embryos require a cold treatment for germination, undried ovules of the same age do not germinate even after a cold treatment. This indicates that mature embryos enclosed in green ovules that germinate viviparously are cold resistant and have not become dormant at the time of culture of siliques. The circumvention by silique culture of a cold treatment and light exposure normally required for germination of isolated seeds of A. thaliana provides new possibilities to study the molecular biology of vivipary and seed germination in this model plant.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Chickpea seed proteins: Modification during germination

Changes in the proteins of chickpea during a 12-day germination period are reported using techniques of gel filtration, DEAE-cellulose chromatography, polyacrylamide gel (PAG) electrophoresis and ultracentrifugation. In the ultracentrifuge, the total proteins of dormant seeds resolve into 3 components which have the sedimentation coefficients of 2.2 S, 6.9 S and 10.3 S respectively. On germination, the presence of fractions of lower sedimentation coefficient indicates possible degradation of these components; in the early stages, the degradation rate of the 7 S fraction is higher, while the 10 S fraction is broken down faster in the later stages. Gel filtration experiments indicate the possibility of degradation of high polymer into intermediary products. Increase in the relative mobility of protein components on PAG and elution constant on DEAE-cellulose chromatographs indicates an increase in the net negative charge of the protein fractions. The accumulation of subunits of the proteins is negligible during the germination period.     

Changes in polyamine concentration during seed germination

Phaseolus mungo seeds 0 to 10 days after germination contained putrescine, spermidine, spermine, cadaverine, agmatine and tyramine. The rate of biosynthesis of total polyamines, proteins and RNA in the developing seeds follows similar profiles, reaching maxima 3 hr from germination. Putrescine, cadaverine, spermidine, spermine and agmatine were the major amines found in Pisum sativum 0–7 days after germination. RNA and proteins seem to follow the same pattern as polyamines during the first 12 hr in the developing pea seeds. RNA reaches a peak at 15 hr and polyamines and proteins peak 24 hr after germination. A rise to total polyamine concentration was also observed in seeds of Tragopogon porrifolius, Zea mays and Triticum aestivum 2–12 hr after germination.     

Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (alpha S1, beta and kappa) are a major class of proteins; however, the dynamic range of concentrations in which the various isoforms of each casein exist presents challenges in their characterization. To study human milk casein phosphoforms, we applied traditional two-dimensional polyacrylamide gel electrophoretic (2-DE) separation combined with matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) tandem mass spectroscopic analysis. The abundant beta-casein was resolved as a train of 6 spots differing in phosphorylation level with 0-5 phosphates attached. To study the less abundant alpha S1-casein, a cysteine-tagging enrichment treatment was used prior to 2-DE. A train of 9 spots with 4.4 < p I < 5.3 were identified as alpha S1-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides ( (27)L-R (51), (69)N-K (98)) consistent with alpha-caseins from various ruminant species. MS/MS analysis of the phosphopeptides released by tryptic digestion enabled identification of the residue-specific order of phosphorylation among the 6 beta-casein and 9 alpha S1-casein phosphoforms. Deamidation of N (47) of alpha S1-casein was also a feature of the MS analysis. This study represents the first comprehensive analysis of the human casein phosphoproteome and reveals a much higher level of phosphorylation than previously recognized. It also highlights the advantages of 2-DE for examining the global pattern of protein phosphoforms and the limitations of attempting to estimate phosphorylation site occupancies from "bottom-up" studies.     

The function of SULTR2;1 sulfate transporter during seed development in Arabidopsis thaliana

SULTR2;1 is a low-affinity sulfate transporter expressed in the vascular tissues of roots and leaves for interorgan transport of sulfate in Arabidopsis thaliana . Transgenic Arabidopsis carrying a fusion gene construct of SULTR2;1 5'-promoter region and β-glucuronidase coding sequence (GUS) demonstrated that within the reproductive tissues, SULTR2;1 is specifically expressed in the bases and veins of siliques and in the funiculus, which connects the seeds and the silique. The antisense suppression of SULTR2;1 mRNA caused decrease of sulfate contents in seeds and of thiol contents both in seeds and leaves, as compared with the wildtype (WT). The effect of antisense suppression of SULTR2;1 on seed sulfur status was determined by introducing a sulfur-indicator construct, p35S::β_SRx3:GUS, which drives the expression of GUS reporter under a chimeric cauliflower mosaic virus 35S promoter containing a triplicate repeat of sulfur-responsive promoter region of soybean β-conglycinin β subunit (β_SRx3). The mature seeds of F1 plants carrying both the SULTR2;1 antisense and p35S::β_SRx3:GUS constructs exhibited significant accumulation of GUS activities on sulfur deficiency, as compared with those carrying only the p35S::β_SRx3:GUS construct in the WT background. These results suggested that SULTR2;1 is involved in controlling translocation of sulfate into developing siliques and may modulate the sulfur status of seeds in A. thaliana .     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.     

Epoetin alpha,epoetin beta and darbepoetin alfa: two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis

Since 1989 recombinant human erythropoietin (rhEPO) has been used as a drug for the correction of anemia, but the misuse of rhEPO as an ergogenic agent among athletes is a widespread doping practice. As a consequence there is a need for developing reference methods for the detection of rhEPO in biological fluids, and to be able to differentiate the recombinant from the natural protein. Recombinant human erythropoietin differs from its natural counterpart in the glycidic part of the molecule. Three different commercial recombinant products Epoetin alpha (Eprex, Janssen Cilag), Epoetin beta (Neorecormon, Roche) and Darbepoetin alfa (Nespo, Dompè) have been used to evaluate the performance of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for the separation of isoforms and the identification of the proteins respectively. All the compounds studied were well separated by means of 2-DE: Epoetin alpha and beta focused in the same isoelectric point region giving rise to six and eight spots respectively, whereas Darbepoetin alfa was found in a more acidic zone with two spots. Results obtained with micro high-performance liquid chromatography-electrospray ionization-time of flight (TOF) MS and matrix-assisted laser desorption/ionization-time of flight MS for the three rhEPOs are reported. These preliminary results suggest that by means of 2-DE and MS it should be possible to reveal the presence of rhEPOs for antidoping purposes.     

Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion

Two-dimensional gel electrophoresis (2-DE), coupled with mass spectroscopy, was used to study seed abortion in Dimocarpus longan Lour. (cv. Minjiao 64-1) by comparing normal and aborted seeds at three developmental stages. More than 1,000 protein spots were reproducibly detected in 2-DE gels, with 43 protein spots being significantly altered in their intensity between normal and aborted seeds at least at one stage. Thirty-five proteins were identified by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS) analysis and protein database searching. Most of the identified proteins were associated with a variety of functions, including energy and metabolism (30%), programed cell death (9%), antioxidative processes (14%), chaperonin (23%), cell division, amino acid metabolism, secondary metabolism, and other functional classes. Furthermore, the expression patterns of HSP70 and cytosolic ascorbate peroxidase (cAPX) were validated by immunoblotting analysis. This study provides a novel, global insight into proteomic differences between normal and aborted seeds in longan. We anticipate that identification of the differentially expressed proteins may lead to a better understanding of the molecular basis for seed abortion in longan.     

Preliminary proteomic analysis of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately

Thiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe2+ or sulphide as energy source. Growth curves for Thiobacillus ferrooxidans have been tested, which show lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Thiobacillus ferrooxidans cultivated with Fe2+ and from 4 to 12 days for Thiobacillus ferrooxidans cultivated with elemental sulphur. Differences of protein patterns of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately were investigated after cultivation at 30 degrees C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ ionization (MALDI)-Mass spectrometry and ESI-MS/MS. From the 17 identified protein spots, 11 spots were found more abundant when growing on elemental sulphur. By contrast 6 protein spots were found decreased at elemental cultivation condition. Among the proteins identified, cytochrome C have been previously identified as necessary elements of electron-transferring pathway for Thiobacillus ferrooxidans to oxidize Fe2+; ATP synthase alpha chain and beta are expressed increased when Thiobacillus ferrooxidans cultivated with Fe2+ as energy source. ATP synthase Beta chain is the catalytic subunit, and ATP synthase alpha chain is a regulatory subunit. The function of ATPase produces ATP from ADP in the presence of a proton gradient across the membrane.