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.

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.

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 ß-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.

Agricultural recovery of a formerly radioactive area: I. Establishment of high-resolution quantitative protein map of mature flax seeds harvested from the remediated Chernobyl area.

In recent years there has been an increasing tendency toward remediation of contaminated areas for agriculture purposes. The study described herein is part of a comprehensive, long-term characterization of crop plants grown in the area formerly contaminated with radioactivity. As a first step, we have established a quantitative map of proteins isolated from mature flax (Linum usitatissimum L.) seeds harvested from plants grown in a remediated plot localized directly in Chernobyl town. Flax was selected because it is a crop of economic and historical importance, despite the relative paucity of molecular resources. We used 2-dimensional electrophoresis followed by tandem mass spectrometry to establish a high-resolution seed proteome map. This approach yielded quantitative information for 318 protein spots. Genomic sequence resources for flax are very limited, leaving us with an "unknown function" annotation for 38% of the proteins analyzed including several that comprise very large spots. In addition to the seed storage proteins, we were able to reliably identify 82 proteins many of which are involved with central metabolism.

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.

Proteomic analysis of mature soybean seeds from the Chernobyl area suggests plant adaptation to the contaminated environment.

The explosion in one of the four reactors of the Chernobyl Nuclear Power Plant (CNPP, Chernobyl) caused the worst nuclear environmental disaster ever seen. Currently, 23 years after the accident, the soil in the close vicinity of CNPP is still significantly contaminated with long-living radioisotopes, such as (137)Cs. Despite this contamination, the plants growing in Chernobyl area were able to adapt to the radioactivity, and survive. The aim of this study was to investigate plant adaptation mechanisms toward permanently increased level of radiation using a quantitative high-throughput proteomics approach. Soybeans of a local variety (Soniachna) were sown in contaminated and control fields in the Chernobyl region. Mature seeds were harvested and the extracted proteins were subjected to two-dimensional gel electrophoresis (2-DE). In total, 9.2% of 698 quantified protein spots on 2-D gel were found to be differentially expressed with a p-value