Proteomic analysis of Chelidonium majus milky sap using two-dimensional gel electrophoresis and tandem mass spectrometry.

Milky sap, a milky-like orange fluid, isolated from the Greater Celandine (Chelidonium majus L.), family Papaveraceae, serves as a rich source of various biologically active substances such as alkaloids, several flavonoids, phenolic acids and proteins. The objective of this study was to separate Ch. majus milky sap extract proteins using two-dimensional gel electrophoresis (2-DE) to demonstrate for the first time the protein composition in the sap and to identify them using liquid chromatography-tandem mass spectrometry analysis (LC-ESI-MS/MS). It was possible to identify 21 proteins, which comprise disease/defence-related, signalling, Krebs cycle, nucleic acid binding and other proteins. The majority of the identified proteins can be linked to direct and indirect stress and defence reactions, e.g. against different pathogens. The specific protein composition of the milky sap suggests an important role of these proteins for the whole plant physiology and development.

Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants of Lolium multiflorum Lam.

Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas of Lolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

Differential influence of bacitracin on plant proteolytic enzyme activities.

The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.

Isozymes in Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids and Ae. kotschyi x S. cereale amphiploids in relation to their parents.

Seven enzymatic systems in F1 Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids, Aegilops kotschyi x S. cereale amphiploids and their parental species (Ae. kotschyi, Ae. biuncialis and S. cereale) were analysed by starch and polyacrylamide gel electrophoresis. Five of them (phosphoglucose isomerase, glutamic oxalacetic transaminase, esterase, acid phosphatase, and diaphorase) were polymorphic and two (malic dehydrogenase and superoxide dismutase) were monomorphic. Several isophorms of phosphoclucose isomerase, esterase, acid phosphatase, and diaphorase were detected in some hybrids and amphiploids, but absent in the parents. The role of regulators, translocations and recombination is discussed in relation to the origin of these new isophorms. Some parental isozymes were absent both in hybrids and amphiploids, probably as a result of the suppression of structural genes in new combinations of the three genomes.

Two-dimensional electrophoresis patterns of total proteins, esterases and acid phosphatases from pollen, seeds and leaves of three cultivars of Helianthus annuus L.

Total proteins, esterases and acid phosphatases from pollen, seeds and leaves of three sunflower cultivars were separated by 2-D electrophoresis. The characteristic peptides for each cultivar were identified. The seeds and pollen of the cultivar Wielkopolski contained 45 and 37 characteristic peptides, respectively, while the seeds and pollen of Coril contained 73 and 35 characteristic peptides. The cultivar Frankasol had the lowest total number of stained peptides in seeds and pollen, and the number of the characteristic peptides was only 61 and 25, respectively. Analyses of esterases and acid phosphatases from young leaves and pollen led to identification of isoenzymes characteristic of the three cultivars. Only for Frankasol no specific acid phosphatase was observed, both in leaves and in pollen.

Maize pollen enzymes after two-dimensional polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate.

Twelve enzymes from mature pollen grains of maize were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2-D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic-oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1-D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2-D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed.