Jiří Šantrůček

Proteins Involved in Distinct Phases of Cold Hardening Process in Frost Resistant Winter Barley (Hordeum vulgare L.) cv Luxor

Winter barley is an economically important cereal crop grown in higher latitudes and altitudes where low temperatures represent an important environmental constraint limiting crop productivity. In this study changes in proteome of leaves and crowns in a frost tolerant winter barley cv. Luxor in relation to short and long term periods of cold followed by a brief frost treatment were studied in order to disclose proteins responsible for the cold hardening process in distinct plant tissues. The mentioned changes have been monitored using two dimensional difference gel electrophoresis (2D-DIGE) with subsequent peptide-mapping protein identification. Regarding approximately 600–700 distinct protein spots detected on 2D gels, there has been found at least a two-fold change after exposure to low temperatures in about 10% of proteins in leaves and 13% of proteins in crowns. Protein and nitrogen metabolic processes have been influenced by low temperature to a similar extent in both tissues while catabolism, carbohydrate metabolism and proteins involved in stress response have been more affected in crowns than in leaves. The range of changes in protein abundance was generally higher in leaves and chloroplast proteins were frequently affected which suggests a priority to protect photosynthetic apparatus. Overall, our data proved existence of slightly different response strategies to low temperature stress in crowns and leaves, i.e., tissues with different biological role. Moreover, there have been found several proteins with large increase in accumulation, e.g., 33 kDa oxygen evolving protein of photosystem II in leaves and “enhanced disease susceptibility 1” in crowns; these proteins might have potential to indicate an enhanced level of frost tolerance in barley.

Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes

Comparative analysis of tobacco EXO70 isoforms reveals their distinct functional properties in tip growth and subcellular localization to several compartments, including specific plasma membrane domains. The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species.

A new allergen family involved in pollen food-associated syndrome: Snakin/gibberellin-regulated proteins

At least two pollen food associated syndromes were described with cypress pollen allergy involving peach and citrus. Snakin/gibberellin regulated proteins are described herein to be the cross-reactive allergens between Cupressus sempervirens pollen and fruit/vegetables.

Trace elements distribution and species fractionation in the wheat (Triticum aestivum) plant.

Abstract The status of 13 trace elements’ (both essential and toxic) was investigated in individual parts of the winter wheat plant(Triticum aestivum) taken during its whole cultivation period. The study includes the determination of total concentrations, portions soluble in 0.02 mol L−1 Tris-HCI buffer solution (pH = 7.5), and the fractionation of soluble species of elements by SEC and ICP/MS. Ligands of trace elements from a low-molecular weight SEC fraction were isolated by affinity chromatography and characterised by MALDI/MS analyses and by amino acids composition. Inhomogeneous accumulation of trace elements was found in the analysed plant tissues. The concentrations of elements are also affected by the maturity of the plants. The distribution of the soluble species of the elements between chromatographic fractions exhibited some regularity in all the samples. Substantial amounts of trace elements are located in a low-molecular weight fraction (< 2 kDa). Only chromatograms of Zn (grain) and Cu (all samples) contain significant medium-molecular and high-molecular weight fractions. Compounds isolated from the low-molecular weight fractions are rich in cystein and dicarboxylic amino acids or their amides. MALDI/MS spectra of these compounds isolated from shoots, straw and grain confirmed the presence of the phytochelatin PC5.

Separation and identification of candidate protein elicitors from the cultivation medium of Leptosphaeria maculans inducing resistance in Brassica napus.

The Dothideomycete Leptosphaeria maculans, a worldwide fungal pathogen of oilseed rape (Brassica napus), secretes a broad spectrum of molecules into the cultivation medium during growth in vitro. Here, candidate elicitor molecules, which induce resistance in B. napus to L. maculans, were identified in the cultivation medium. The elicitation activity was indicated by increased transcription of pathogenesis‐related gene 1 (PR1) and enhanced resistance of B. napus plants to the invasion of L. maculans. The elicitation activity was significantly lowered when the cultivation medium was heated to 80°C. Active components were further characterized by specific cleavage with the proteolytic enzymes trypsin and proteinase K and with glycosidases α‐amylase and β‐glucanase. The elicitor activity was eliminated by proteolytic digestion while glycosidases had no effect. The filtered medium was fractionated by either ion‐exchange chromatography or isoelectric focusing. Mass spectrometry analysis of the most active fractions obtained by both separation procedures revealed predominantly enzymes that can be involved in the degradation of plant cell wall polysaccharides. This is the first study searching for L. maculans‐specific secreted elicitors with a potential to be used as defense‐activating agents in the protection of B. napus against L. maculans in agriculture.

Isolation of Ligands of Trace Metals from Plant Samples by Immobilized Metal Affinity Chromatography

Abstract Isolation of ligands of trace elements from plants by affinity chromatography with a metal immobilised on iminodiacetate resin has been investigated. To simulate various types of bioligands the following compounds were tested: phytic acid, peptides containing Cys and peptides containing Asp. Optimal conditions allowing isolation of the peptides with good efficiency comprise the use of Ga3+ ion, sample adsorption at pH = 4 and elution of the compounds by 0.3 mol/l NH3. Phytic acid was bound too tightly and was not eluted. The procedure was used for purification of extracts of rye flour. The purified sample is suitable for analysis by MALDI-MS.

The effect of Fusarium culmorum infection and deoxynivalenol (DON) application on proteome response in barley cultivars Chevron and Pedant

Fusarium head blight (FHB) disease adversely affects grain quality and final yield in small-grain cereals including barley. In the present study, the effect of an artificial infection with Fusarium culmorum and an application of deoxynivalenol (DON) on barley spikes of cultivars Chevron and Pedant during flowering was investigated at grain mid-dough stage (BBCH 73) 10days after pathogen inoculation (10 dai). Proteomic analysis using a two-dimensional differential gel electrophoresis (2D-DIGE) technique coupled with LC-MS/MS investigated 98 protein spots revealing quantitative or qualitative differences between the experimental variants. Protein functional annotation of 93 identified protein spots revealed that most affected functional groups represent storage proteins (globulins, hordeins), followed by proteins involved in carbohydrate metabolism (α-amylase inhibitor, β-amylase, glycolytic enzymes), amino acid metabolism (aminotransferases), defence response (chitinase, xylanase inhibitor, serpins, SGT1, universal stress protein USP), protein folding (chaperones, chaperonins), redox metabolism (ascorbate-glutathione cycle), and proteasome-dependent protein degradation. The obtained results indicate adverse effects of infection on plant proteome as well as an active plant response to pathogen as shown by enhanced levels of several inhibitors of pathogen-produced degradation enzymes (α-amylase inhibitor, xylanase inhibitor, serpins), chaperones, and other stress-related proteins (SGT1, USP). Genotypic differences were found in hordein abundance between Chevron and Pedant.