Katarína Klubicová

Mass Spectrometry-Based Analysis of Proteomic Changes in the Root Tips of Flooded Soybean Seedlings

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.

Proteomics analysis of flax grown in Chernobyl area suggests limited effect of contaminated environment on seed proteome.

The accident at the Chernobyl Nuclear Power Plant (CNPP) on April 26, 1986 is the most serious nuclear disaster in human history. Surprisingly, while the area proximal to the CNPP remains substantially contaminated with long-lived radioisotopes including (90)Sr and (137)Cs, the local ecosystem has been able to adapt. To evaluate plant adaptation, seeds of a local flax (Linum usitatissimum) variety Kyivskyi were sown in radio-contaminated and control fields of the Chernobyl region. A total protein fraction was isolated from mature seeds, and analyzed using 2-dimensional electrophoresis combined with tandem-mass spectrometry. Interestingly, growth of the plants in the radio-contaminated environment had little effect on proteome and only 35 protein spots differed in abundance (p-value of ≤0.05) out of 720 protein spots that were quantified for seeds harvested from both radio-contaminated and control fields. Of the 35 differentially abundant spots, 28 proteins were identified using state-of-the-art MS(E) method. Based on the observed changes, the proteome of seeds from plants grown in radio-contaminated soil display minor adjustments to multiple signaling pathways.

Soybeans Grown in the Chernobyl Area Produce Fertile Seeds that Have Increased Heavy Metal Resistance and Modified Carbon Metabolism

Plants grow and reproduce in the radioactive Chernobyl area, however there has been no comprehensive characterization of these activities. Herein we report that life in this radioactive environment has led to alteration of the developing soybean seed proteome in a specific way that resulted in the production of fertile seeds with low levels of oil and β-conglycinin seed storage proteins. Soybean seeds were harvested at four, five, and six weeks after flowering, and at maturity from plants grown in either non-radioactive or radioactive plots in the Chernobyl area. The abundance of 211 proteins was determined. The results confirmed previous data indicating that alterations in the proteome include adaptation to heavy metal stress and mobilization of seed storage proteins. The results also suggest that there have been adjustments to carbon metabolism in the cytoplasm and plastids, increased activity of the tricarboxylic acid cycle, and decreased condensation of malonyl-acyl carrier protein during fatty acid biosynthesis.

Agricultural recovery of a formerly radioactive area: II. Systematic proteomic characterization of flax seed development in the remediated Chernobyl area

Molecular characterization of crop plants grown in remediated, formerly radioactive, areas could establish a framework for future agricultural use of these areas. Recently, we have established a quantitative reference map for mature flax seed proteins (Linum usitatissimum L.) harvested from a remediated plot in Chernobyl town. Herein we describe results from our ongoing studies of this subject, and provide a proteomics-based characterization of developing flax seeds harvested from same field. A quantitative approach, based on 2-dimensional electrophoresis (2-DE) and tandem mass spectrometry, yielded expression profiles for 379 2-DE spots through seed development. Despite the paucity of genomic resources for flax, the identity for 102 proteins was reliably determined. These proteins were sorted into 11 metabolic functional classes. Proteins of unknown function comprise the largest group, and displayed a pattern of decreased abundance throughout seed development. Analysis of the composite expression profiles for metabolic protein classes revealed specific expression patterns during seed development. For example, there was an overall decrease in abundance of the glycolytic enzymes during seed development.

Radioactive Chernobyl Environment Has Produced High-Oil Flax Seeds That Show Proteome Alterations Related to Carbon Metabolism during Seed Development

Starting in 2007, we have grown soybean (Glycine max [L.] Merr. variety Soniachna) and flax (Linum usitatissimum, L. variety Kyivskyi) in the radio-contaminated Chernobyl area and analyzed the seed proteomes. In the second-generation flax seeds, we detected a 12% increase in oil content. To characterize the bases for this increase, seed development has been studied. Flax seeds were harvested in biological triplicate at 2, 4, and 6 weeks after flowering and at maturity from plants grown in nonradioactive and radio-contaminated plots in the Chernobyl area for two generations. Quantitative proteomic analyses based on 2-D gel electrophoresis (2-DE) allowed us to establish developmental profiles for 199 2-DE spots in both plots, out of which 79 were reliably identified by tandem mass spectrometry. The data suggest a statistically significant increased abundance of proteins associated with pyruvate biosynthesis via cytoplasmic glycolysis, L-malate decarboxylation, isocitrate dehydrogenation, and ethanol oxidation to acetaldehyde in early stages of seed development. This was followed by statistically significant increased abundance of ketoacyl-[acylcarrier protein] synthase I related to condensation of malonyl-ACP with elongating fatty acid chains. On the basis of these and previous data, we propose a preliminary model for plant adaptation to growth in a radio-contaminated environment. One aspect of the model suggests that changes in carbon assimilation and fatty acid biosynthesis are an integral part of plant adaptation.

Do Cupins Have a Function Beyond Being Seed Storage Proteins

Plants continue to flourish around the site of the Chernobyl Nuclear Power Plant disaster. The ability of plants to transcend the radio-contaminated environment was not anticipated and is not well understood. The aim of this study was to evaluate the proteome of flax (Linum usitatissimum L.) during seed filling by plants grown for a third generation near Chernobyl. For this purpose, seeds were harvested at 2, 4, and 6 weeks after flowering and at maturity, from plants grown in either non-radioactive or radio-contaminated experimental fields. Total proteins were extracted and the two-dimensional gel electrophoresis (2-DE) patterns analyzed. This approach established paired abundance profiles for 130 2-DE spots, e.g., profiles for the same spot across seed filling in non-radioactive and radio-contaminated experimental fields. Based on Analysis of Variance (ANOVA) followed by sequential Bonferroni correction, eight of the paired abundance profiles were discordant. Results from tandem mass spectrometry show that four 2-DE spots are discordant because they contain fragments of the cupin superfamily-proteins. Most of the fragments were derived from the N-terminal half of native cupins. Revisiting previously published data, it was found that cupin-fragments were also involved with discordance in paired abundance profiles of second generation flax seeds. Based on these observations we present an updated working model for the growth and reproductive success of flax in a radio-contaminated Chernobyl environment. This model suggests that the increased abundance of cupin fragments or isoforms and monomers contributes to the successful growth and reproduction of flax in a radio-contaminated environment.

Seeds in Chernobyl: the database on proteome response on radioactive environment

Two serious nuclear accidents during the last quarter century (Chernobyl, 1986 and Fukushima, 2011) contaminated large agricultural areas with radioactivity. The database “Seeds in Chernobyl” (http://www.chernobylproteomics.sav.sk) contains the information about the abundances of hundreds of proteins from on-going investigation of mature and developing seed harvested from plants grown in radioactive Chernobyl area. This database provides a useful source of information concerning the response of the seed proteome to permanently increased level of ionizing radiation in a user-friendly format.

Systems biology is an efficient tool for investigation of low-dose chronic irradiation influence on plants in the Chernobyl zone

The paper discusses different methodological approaches to the study of transgenerational alterations of metabolic pathways in soybean and flax seeds in the process of adaptation to chronic irradiation in the Chernobyl alienation zone. A combination of general biological methods and novel approaches, such as genomics, proteomics, cytogenetics, and mutagenesis, allows researchers to analyze an organism’s systemic response and identify the latent chronic irradiation effects in plants from the Chernobyl zone. The proteomic approaches are especially efficient, since they range from the identification of changes in abundance and folding of individual proteins to the characterization of posttranslational modifications, trends of qualitative changes during seed maturation, or protein-protein interactions during plant growth and development under permanent impacts of stress factors. The application of proteomics opens new horizons in the understanding of the hidden mechanisms behind the impact of chronic low-dose radiation on living cells and makes it possible to visualize metabolic network alterations regardless of their transcriptional, translational, or epigenetic nature.

Proteomic insights into intra- and intercellular plant-bacteria symbiotic association during root nodule formation

Over the last several decades, there have been a large number of studies done on the all aspects of legumes and bacteria which participate in nitrogen-fixing symbiosis. The analysis of legume–bacteria interaction is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. Bacteria regulate their quorum-sensing genes to enhance their ability to induce conjugation of plasmids and symbiotic islands, and various protein secretion mechanisms; that can stimulate a collection of chain reactions including species-specific combinations of plant-secretion isoflavonoids, complicated calcium signaling pathways and autoregulation of nodulation mechanisms. Quorum-sensing systems are introduced by the intra- and intercellular organization of gene products lead to protein–protein interactions or targeting of proteins to specific cellular structures. In this study, an attempt has been made to review significant contributions related to nodule formation and development and their impacts on cell proteome for better understanding of plant–bacterium interaction mechanism at protein level. This review would not only provide new insights into the plant–bacteria symbiosis response mechanisms but would also highlights the importance of studying changes in protein abundance inside and outside of cells in response to symbiosis. Furthermore, the application to agriculture program of plant–bacteria interaction will be discussed.

Insights into the early stage of Pinus nigra Arn. somatic embryogenesis using discovery proteomics

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Arn. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryogenic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing. BIOLOGICAL SIGNIFICANCE Somatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Arn. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues.