Radioactive Chernobyl Environment Has Produced High-Oil Flax

Article pubs.acs.org/jpr

Radioactive Chernobyl Environment Has Produced High-Oil Flax Seeds That Show Proteome Alterations Related to Carbon Metabolism during Seed Development Katarína Klubicová,† Maksym Danchenko,†,§ Ludovit Skultety,∥,⊥,# Valentyna V. Berezhna,§ Namik M. Rashydov,§ and Martin Hajduch*,†,‡,∥ †

Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P.O. Box 39A, Nitra 95007, Slovakia Institute of Chemistry, Center of Excellence for White-Green Biotechnology, Slovak Academy of Sciences, Trieda Andreja Hlinku 2, Nitra 94976, Slovakia § Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv 03680, Ukraine ∥ Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 84505, Slovakia ⊥ Center for Molecular Medicine, Slovak Academy of Sciences, Vlárska 3-7, Bratislava 83101, Slovakia # Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, Prague 11720, Czech Republic ‡

S Supporting Information *

ABSTRACT: 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. KEYWORDS: radioactivity, seed, development, abiotic stress, fatty acids, flax, two-dimensional electrophoresis

1. INTRODUCTION We use the Chernobyl area as an open field laboratory to observe alterations in the soybean and flax seed proteome during adaptation toward a radioactive environment. The research is based on a 2-D electrophoresis plus tandem mass spectrometry strategy. Data acquired during the course of these experiments have been deposited on an online database available at www.chernobylproteomics.sav.sk1 after publication. The results from the analysis of the first generation of mature soybean seeds suggested that adaptation to heavy metal stress, protection against radiation damage, and mobilization of seed storage proteins (SSPs) are important to recovery mechanisms in the radio-contaminated Chernobyl area.2 Results from subsequent second-generation soybean seed analyses confirmed these data and more specifically suggested adjustments in both © 2013 American Chemical Society

cytoplasmic and plastidial carbon metabolism, increased activity of the tricarboxylic acid cycle, and decreased condensation of malonyl-acyl carrier protein during fatty acid (FA) biosynthesis.3 In contrast, the flax seed proteome seems to be less affected by plant growth in the radio-contaminated environment. The analysis of first-generation mature flax seeds harvested from the Chernobyl area identified only minor adjustments in protein abundance.4 Typically, flax seeds contain 41% oil, 20% protein, and 28% dietary fiber.5 The main obstacle to proteomic Special Issue: Agricultural and Environmental Proteomics Received: June 5, 2013 Published: October 11, 2013 4799

dx.doi.org/10.1021/pr400528m | J. Proteome Res. 2013, 12, 4799−4806

Journal of Proteome Research

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analyses of flax had been the limited genome resources. This situation recently improved with the sequences for 59 626 unigenes becoming available6 along with the whole genome assembly from shotgun sequencing.7 Moreover, large-scale analysis of expressed sequences in various tissues of flax provided novel information apart from the unigene data set,8 and new genes involved in flax seed maturation were identified.9 Importantly, proteomic analysis of seven stages of flax seed development confirmed the importance of lipid biosynthesis during embryogenesis and seed filling.10 Previously, we analyzed mature flax seeds harvested in 2008 from a remediated area of Chernobyl town.11,12 These experiments mapped seed proteins of flax grown in the area formerly contaminated with radioactivity. Herein we utilize the recent developments in flax genome research and complement our previous data obtained from radio-contaminated environment with comparative quantitative proteomics analysis of flax seed development in the Chernobyl area.

2.3. Plant Material

Flax (Linum usitatissimum L.; variety Kyivskyi) was grown in the Chernobyl fields beginning in 2007 (Figure S1 in the Supporting Information). During the 2008 growing season, the second generation of seeds was harvested at 2, 4, and 6 weeks after flowering (WAF) and maturity (Figure 1) from radio-

2. EXPERIMENTAL PROCEDURES 2.1. Radioactivity Measurements

The measurements of 90Sr radioactivity in harvested seeds were carried out using the gravimetric method for β-radiometry and a low-background UMF-1500 M instrument, as previously described.13 Measurements of γ radioactivity using a semiconductivity coaxial detector of superpure Germanium, with an energetically resolved 1332.5 keV peak of 60Co, were performed, as we recently described.3 The transfer coefficient (TC) was calculated by dividing plant radioactivity by soil radioactivity (kBq·kg−1).

Figure 1. Characterization of mature seeds grown in nonradioactive and radioactive fields in the Chernobyl area. (A) Total oil content in mature flaxseeds. Standard deviations are shown as error bars. (B) Uptake of radionuclides 137Cs and 90Sr into mature flax seeds expressed as transfer coeficient (TC).

contaminated and nonradioactive fields (Figure S1 in the Supporting Information). To ensure reliable interpretations of the data, we have performed three independent seed harvests (Table S2 in the Supporting Information).

2.2. Field and Soil Characterization

Two experimental fields were established in the Chernobyl area (Figure S1 in the Supporting Information). The radiocontaminated field is located 5 km from CNPP near the village Chistogalovka (soil radioactivity 20 650 ± 1050 Bq·kg−1 of 137 Cs and 5180 ± 550 Bq·kg−1 of 90Sr). Nonradioactive field (control) is located directly in Chernobyl town (soil radioactivity of 1414 ± 71 Bq·kg−1 of 137Cs and 550 ± 55 Bq·kg−1 of 90 Sr) (Table 1). Abiotic factors, such as drought and nutrition,

2.4. Total Oil Extraction

Total oil was extracted using a Soxhlet extractor and diethylether, as previously described,14 in three independent experiments. In brief, 1 g of seeds was homogenized using a mortar and pestle and dried at 105 °C for 3 h. Then, 250 mL of ether was added and oil was extracted for 16 h. After extraction, samples were air-dried until residual ether could not be detected. Oil content was determined gravimetrically.

Table 1. Radioactivity of Radioactive and Control Chernobyl Fields and Mature Seeds Harvested from Both Fields radioactivity Bg·kg 137

Radio-Contaminated Field soil mature flaxseeds Nonradioactive Field soil mature flaxseeds

Cs

2.5. Protein Extraction

‑1

Total protein was isolated from each independent seed harvest using a phenol-based protocol15 with minor modifications.4 The number of seeds used for independent protein extractions are shown in Table S2 in the Supporting Information. In brief, seeds were homogenized with (50% [v/v] phenol, 0.45 M sucrose, 5 mM EDTA, 0.2% [v/v] 2-mercaptoethanol, 50 mM Tris-HCl, pH 8.8), then proteins precipitated with 5 volumes of ice cold 0.1 M ammonium acetate in 100% methanol, and washed successively with 0.1 M ammonium acetate in 100% methanol, ice cold acetone, and 70% ethanol. Protein pellets were dried under reduced pressure, then solubilized in 0.5 mL of immobilized pH gradient (IPG) buffer containing 8 M urea, 2 M thiourea, 2% CHAPS, 2% Triton X-100, and 50 mM DTT, with gentle agitation for 30 min at 4 °C. Protein quantification was performed using the Bradford Reagent (Sigma-Aldrich, Saint Louis, MO) with bovine serum albumin as the standard.

90

Sr

20650 ± 1050 780 ± 39

5180 ± 550 3550 ± 360

1414 ± 71 8±5

550 ± 55 90 ± 16

are similar in both fields. Soil in the radio-contaminated field have pH of 5.6 and soil in nonradioactive field have pH of 6.6 (Table S1-A in the Supporting Information). In both locations, aleurite (silt) and pelitic soil contents range from 20 to 30%. The soils are sod-podzolic with a loamy-sand texture derived from sandy fluvio-glacial deposits, contain 12% clay and 2% organic material, and have an electric conductivity of 0.20 dS· m−1. In total, 22.9% of soil particles have size between 0.1 and 0.2 mm (Table S1-B in the Supporting Information).

2.6. Protein Separation

Protein preparations from independent seed harvests were analyzed by 2-D electrophoresis (2-DE). For the analysis, 700 4800



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Figure 2. Experimental workflow. Proteins were isolated from seeds grown in nonradioactive and radioactive Chernobyl area and harvested at 2, 4, and 6 weeks after flowering (WAF) and at maturity. Proteins were separated by 2D electrophoresis, the gels were imaged, and images of each data set were matched to the reference (pooled) gels (i.e., seed from nonradioactive and radioactive Chernobyl areas). The 79 proteins were identified using tandem mass spectrometry.

μg of proteins in IPG buffer was loaded onto IPG strips (pH 5 to 8; 17 cm; Bio-Rad, Hercules, CA) and placed into an isoelectric focusing (IEF) unit (Protean IEF Cell, Bio-Rad, Hercules, CA). After IEF, excess mineral oil was removed and the strips were equilibrated for 15 min in 4 mL of reduction solution (2% DTT in equilibration buffer containing 1.5 M Tris, pH 6.8, 6 M urea, 30% glycerol, and 5% SDS) and for 15 min in 4 mL of 2.5% iodoacetamide in equilibration buffer. The strips were rinsed in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS), placed on top of an SDS gel (12% separation gel), and overlaid with sealing solution (0.5% (v/w) agarose in running buffer with 0.002% bromphenol blue as the tracking dye). Second dimension separation was carried out using a Protean II xi Cell (Bio-Rad, Hercules, CA), at 10 mA current for ∼14 h until the dye migrated off the gel. All samples were analyzed in biological triplicate.

Only 2-DE spots that satisfied the following criteria were included in the analysis; a 2-DE spot must be present in both data sets (i.e., from radioactive and nonradioactive fields), and in each data set, a 2-DE spot must be present in at least two biological replicates and three or more developmental stages. A total of 199 2-DE spots satisfied these thresholds (Figure 2). The volumes of all spots that satisfied these criteria were normalized to compensate for small differences in sample loading or gel staining (Table S3 in the Supporting Information). The p value for each 2-DE spot in all seed stages was calculated in Excel to determine statistically significant difference in protein abundance profiles between radio-contaminated and control field (Table S4 in the Supporting Information). 2.9. Mass Spectrometry

Excised 2-DE gel plugs were washed with 500 μL of 50% acetonitrile in 50 mM ammonium bicarbonate, dehydrated in 100% acetonitrile, and rehydrated in digestion solution that contained modified sequencing grade trypsin (Promega). After overnight digestion at 37 °C, the tryptic peptides were extracted, transferred to a microplate, lyophilized, and stored at −80 °C until use. Digested proteins were subjected to dataindependent MSE analysis, which uses alternate scans at low and high collision energies,16 as we recently described.3 After resuspension, tryptic peptides were injected onto the column (nanoAcquity UPLC column BEH 130 C18, 100 μm × 150 mm, 1.7 μm particle size) at a flow rate of 350 nL/min that was connected to the PicoTip emitters (New Objective, USA) and mounted into the nanospray source (3.4 kV at 70 °C) of the quadrupole time-of-flight mass spectrometer (Q-TOF Premier, Waters, U.K.). An acetonitrile gradient at a flow rate of 350 nL/ min (10−45% B in 40 min; A = water with 0.1% formic acid, B = acetonitrile containing 0.1% formic acid) was used to separate peptides. Spectral acquisition scan rates were set to 1 s with a 0.05 s interscan delay. The external mass calibrant Glu-1fibrinopeptide B (500 fmol/mL) was infused via the Lock-mass source at a flow rate of 500 nL/min and sampled every 30 s. A constant collision energy of 20 eV was used to collect calibrant

2.7. Gel Staining and Image Analysis

After completion of the 2-DE step, the gels were washed three times for 15 min in deionized H2O and stained overnight in colloidal Coomassie (20% ethanol, 1.6% phosphoric acid, 8% ammonium sulfate, 0.08% Coomassie Brilliant Blue G-250). The 2-DE gels were digitalized using a GS-800 calibrated densitometer (Bio-Rad, Hercules, CA) at 300 dpi and a 16 bit grayscale. Digitalized gels were analyzed using ImageMaster software 4.9 (GE Healthcare, Uppsala, Sweden) that includes spot detection, quantification, background subtraction, and spot matching between multiple gels. The reference gel was created by pooling equal amounts of protein (175 mg) from each developmental stage. 2.8. Establishing Protein Abundance Profiles

Protein abundance profiles during flax seed development were established as we previously described.3 In brief, gels of each developmental stage were matched individually to the reference gel (pooled sample from all investigated stages) in biological triplicate using ImageMaster software. All matched spots were grouped into subclasses and developmental profiles were established (Figure 2). 4801



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Mature flax seeds showed small amounts of 137Cs (Table 1, Figure 1), in agreement with the measurements from firstgeneration seeds harvested from the radioactive Chernobyl area.4 However, the accumulation of 90Sr was higher than that of 137Cs (Table 1, Figure 1). Notably, radionuclide accumulation in mature flax seeds (780 ± 39 Bq·kg−1 of 137Cs; 3550 ± 360 Bq·kg−1of 90Sr) was significantly lower than uptake of these radionuclides into soybeans (2130 ± 207 Bq·kg−1 of 137Cs, 11 840 ± 1800 Bq·kg−1 of 90Sr) that were grown in the same fields.3

data. Alternating, low (MS, 3 eV) and elevated energy collision energy modes16 were used to collect MSE data. In elevated energy mode (MSE), the collision energy was ramped from 20 to 38 eV during each integration. 2.10. Data Processing, Protein Identification, And Subcellular Localization

The MSE data were processed using the IDENTITYE search algorithm within the ProteinLynx Global Server v. 2.4 (PLGS, Waters, U.K.) and searched against an “in house” assembled database that contained 44 304 nonredundant Linum sequences. This database was created in October 2012 from 802, 948, and 43 236 Linum sequences downloaded from UniProt, NCBI, and Phytozome,7 respectively. Additionally, common contaminants keratin (UniProt Q16195) and trypsin (Uniprot P07477) were added to this database. For initial correlation of a precursor (MS) and possible fragment ions (MS/MS), the time alignment was applied. Search parameters included (i) mass accuracy 50 ppm for precursor ions and 0.1 Da for product ions, (ii) a minimum of three consecutive product ion matches per peptide, and (iii) a minimum of seven total product ion matches per protein. The maximum false positive rate against the randomized forward database was set to 4%. Only one missed tryptic cleavage site was allowed during the search. Fixed modifications included carbamidomethylation of Cys. Variable modifications included oxidation of Met, deamidation of Asn and Gln, and dehydration of Ser and Thr. To analyze all MSE data, we calculated a PLGS score during the database search using a Monte Carlo algorithm. The PLGS score is a statistical measure of accuracy of assignation. After the database search, the additional thresholds were applied to accept protein identification; a minimum of two peptides matched to the protein sequence and a PLGS score greater than 50. To specify the predicted subcellular localization of identified proteins, we used the algorithms TargetP (http:// www.cbs.dtu.dk/services/TargetP/), iPSORT (http://ipsort. hgc.jp/), and Pretodar (http://urgi.versailles.inra.fr/predotar/ predotar.html). The subcellular localization of a particular protein was accepted when confirmed by at least two algorithms.

3.2. Changes in Abundance of 79 Proteins Were Determined during Seed Development

To allow direct comparison with our previous data, we followed the experimental design developed for soybean3 exactly. In brief, total proteins were isolated from developing and mature seeds harvested from nonradioactive and radioactive fields located in the Chernobyl area (Figure S1 in the Supporting Information). Proteins were resolved by 2-DE (Figure S2 in the Supporting Information), stained, and analyzed with ImageMaster software (Figure 2). A total of 199 spots that satisfied these criteria were taken for protein identification (Table S3, Figure S3 in the Supporting Information). Mass spectrometry provided the identity for 79 proteins, 49 of which are nonredundant (Table S5 in the Supporting Information). These proteins were classified into nine functional categories according to Bevan et al.17 with modification (Table S5 in the Supporting Information, Figure 3).18 Thirty five proteins were

Figure 3. Classification of 79 identified proteins. Proteins identified by data-independent MSE method were classified according to Bevan et al.,17 as modified by Miernyk et al.18 The most represented classes were protein destination and storage, energy, and disease/defense proteins.

3. RESULTS 3.1. Flax Seeds from the Radioactive Chernobyl Area Contained More Oil and Accumulated Less Radioactivity than Soybeans

associated with destination and storage. The second most abundant functional group included 15 proteins associated with energy, followed by 8 disease/defense proteins, 8 proteins of unknown function, 6 proteins associated with primary metabolism, and 4 cell growth proteins (Figure 3).

Since 2007, flax plants have been grown in both radiocontaminated and nonradioactive experimental fields established in the Chernobyl area (Figure S1 in the Supporting Information). During the 2008 season, the second generation of developing and mature flax seeds was harvested from both experimental fields. Mature flax seeds harvested from a nonradioactive Chernobyl area control field contained 39.7 ± 1.5% oil, while seeds from radio-contaminated Chernobyl area fields contained 44.8 ± 1.4% oil (Table 2, Figure 1).

3.3. Developmental Profiles Characterized Proteins during Flax Seed Development in Chernobyl

Developmental profiles were established for all 79 proteins in radio-contaminated and nonradioactive Chernobyl areas, as we previously described.3 The p value was calculated for the abundances of these proteins in nonradioactive and radioactive Chernobyl fields (Table S4 in the Supporting Information). Statistically significant difference was considered only when generated p value was equal to or less than 0.05 (Table S4 in the Supporting Information). For instance, the abundance of the chloroplast proteins, 3-ketoacyl−acyl carrier protein synthase I (spot #237 in nonradioactive fields/spot #234 in radio-contaminated field, and large subunit of ribulose 1,5bisphosphate carboxylase oxygenase (RuBisCO; spot #165/

Table 2. Total Oil Content in Mature Flax Seeds Harvested from Chernobyl Fields nonradioactive field radio-contaminated field

dry mass (%)

oil (% of mass)

92.20 ± 1.3 91.36 ± 1.2

39.74 ± 1.45 44.83 ± 1.42 4802



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#181) were statistically significantly altered during early stages of seed development in the radio-contaminated area (Table S4 in the Supporting Information). This developmental pattern was also followed by phosphoglycerate kinase (spot #259/ #252), enolase (spot #146/#160), lactate/malate dehydrogenase family protein (spot #318/#328), alcohol dehydrogenase 1 (spot #282/#280), cytoplasmic NADP+-dependent isocitrate dehydrogenase (spot #226/#225), and NADH:cytochrome B5 reductase 1 (spot #60/#68) (Table S4 in the Supporting Information). The abundance of some SSP subunits also differed during development between radio-contaminated and nonradioactive fields. For instance, 351/357, 471/472, and 484/486 were more abundant in seeds harvested from the radio-contaminated area (Table S4 in the Supporting Information). All data from this study are also available in user-friendly format at http://www.chernobylproteomics.sav. sk/results/20.

beyond transcription, as shown during the investigation of concurrence between protein/transcript pairs during Arabidopsis thaliana seed development.22 Moreover, protein abundance does not reflect pathway flux due to kinetic properties or branching and circular nature of metabolic pathways.23,24 Despite these limitations, the present results provide insight into proteome-level alterations associated with FA biosynthesis detected during flax seed development in the radio-contaminated Chernobyl area. 4.1. Abundance of Proteins Associated with Carbon Metabolism Increased at Early Stages of Flax Seed Development in Radio-Contaminated Environment

Photosynthesis provides carbon for de novo FA biosynthesis. In this regard, the present study identified two 2-DE spots (161/ 180, 165/181) as the large subunit of RuBisCO, a key enzyme of photosynthesis (Figure 5). Spot 165/181 statistically significantly increased in abundance in early stages of flax seed development in radio-contaminated Chernobyl area (Table S4 in the Supporting Information), which suggests increased activity of photosynthetic accumulation during flax

3.4. Proteins Associated with Metabolism and Energy Were Significantly More Abundant in Early Stages of Seed Development in Radio-Contaminated Chernobyl Area Fields

Composite developmental profiles were established as previously described.3,19 The purpose of these profiles is to view the overall abundance of proteins of a specific metabolic class (Figure 4). While the abundance of some SSP was altered,

Figure 4. Composite expression profiles for functional classes metabolism, energy, and destination and storage. Composite profiles were set up by calculating the sum of abundance profiles for individual proteins classified within each functional class during flaxseed development (2, 4, 6 WAF, and mature seeds) in radioactive and nonradioactive Chernobyl fields. The Y axis shows the relative volume (V). The number of proteins within each composite protein abundance profile is shown.

the differences were not significant, and overall abundances of all 35 SSP were similar during development in the both Chernobyl areas. However, overall abundances of proteins associated with metabolism and energy significantly increased in early stages of seed development in radio-contaminated fields (Figure 4).

Figure 5. Schematic view of metabolic pathways connected with FA biosynthesis and energy. Graphs show relative volumes of protein spots during development. Proteins were linked to metabolic pathways. Dashed arrows are used when no protein was detected. Highlighted abundance profiles indicate statistical significant difference between radio-contaminated and nonradioactive fields (Table S4 in the Supporting Information). * is used when p ≤ 0.05 and ** is used when p ≤ 0.01. Abbreviations for metabolites: ACP, acyl carrier protein; ADP, adenosine diphosphate; ATP, adenosine triphosphate; 1,3 bis PGA, 1,3 bis phosphoglyceric acid; 2-PGA, 2 phosphoglyceric acid; 3-PGA, 3 phosphoglyceric acid; DHAP, dihydroxyacetone phosphate; G-3-P, glyceraldehyde 3-phosphate; Glc-1-P, glucose 1 phosphate; Glc-6-P, glucose 6 phosphate; PEP, phosphoenolpyruvate; UDP-Glc, UDP-glucose; FA, fatty acid; NADP, nicotinamide adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; UDP, uridine diphosphate; UTP, uridine triphosphate.

4. DISCUSSION The aim of this study was to use a proteomic approach to investigate changes in the abundance of enzymes involved in stress responses. An unexpected observation was the increased oil content in mature seeds harvested from the second generation of flax grown in the radio-contaminated Chernobyl area (Figure 1). Proteomics can be a useful tool for the study of oil synthesis and accumulation. For instance, it was shown that carbon mobilization from glucose to coenzyme A and its acyl derivative is a collaboration between the cytoplasm and plastids during oilseed rape seed development.20 However, to correlate proteomic data with phenotype is difficult.21 One reason is that temporal control of many enzymes and pathways extends 4803



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ase 1 (spot 282/280), which catalyzes the oxidation of ethanol to acetaldehyde (Figure 5). Acetaldehyde can then be decarboxylated to pyruvic acid by pyruvate decarboxylase.

seed development in a radio-contaminated environment. Association of RuBisCO with abiotic stress response was previously shown. For instance, RuBisCO was proposed as a metabolic indicator of drought tolerance25 and increased in abundance during cadmium stress.26 This study detected increased abundance of proteins associated with pyruvate biosynthesis through cytoplasmic glycolysis, L-malate decarboxylation, isocitrate dehydrogenation, and ethanol oxidation to acetaldehyde in the early stages of seed development (Figure 5). Pyruvate is the most effective substrate for FA synthesis, as revealed by 14C-labeling.27 Disruption of pyruvate kinase, which catalyzes conversion of phosphoenolpyruvate to pyruvate, resulted in a 60% reduction of oil content in the seed of the reference eudicot plant A. thaliana.28 Pyruvate also provides reducing equivalents and energy through malate synthesis, Krebs cycle, or pentose phosphate pathway.29,30 Thus, increased abundance of enzymes associated with pyruvate synthesis might not always result in increased oil production. It was shown that transcripts of genes involved in the conversion of sucrose to pyruvate were not related to oil variation in oil palm.31 Additionally, transcript levels of most of cytoplasmic glycolytic enzymes were found at similar levels when oil palm was compared with closely related date palm.32 In this study, phosphoglycerate kinase (PGK, spot 259/252), and enolase (spot 146/160) were statistically significantly more abundant in early stages of flax seed development in the radiocontaminated Chernobyl area (Tables S3 and S4 in the Supporting Information, Figure 5). The association of these glycolytic enzymes with abiotic stress was previously described. Phosphoglycerate kinase increased in abundance also in the first generation of mature flax seeds harvested from the radiocontaminated Chernobyl experimental field in 2007.4 Phosphoglycerate kinase was found to be differentially abundant during flooding stress,33 salt stress,34 or ozone stress.35 Enolase was suggested as one of the key proteins for chilling injury,36 drought,37 and salt stress.38 Apart from glycolytic enzymes, NADP-malic enzyme 1 (NADP-ME), which catalyzes the oxidative decarboxylation of L-malate to pyruvate, was also found to be statistically significantly more abundant at early stages of flax seed development in radio-contaminated area (Table S4 in the Supporting Information, Figure 5). Previously it was shown that NADP-ME is associated with drought,39 high salt,40 and osmotic41 stresses. In the cytoplasm, production of 2-oxoglutarate (α-ketoglutarate) from isocitrate is catalyzed by NADP-dependent isocitrate dehydrogenase (NADP-ICDH), a key enzyme in lipid metabolism.42 The production of pyruvate from L-alanine is catalyzed by alanine-2-oxoglutarate aminotransferase 2 (Ala AT) and NADP-ICDH (spot 226/225), which was found to be statistically increased in early stages of seed development in the radio-contaminated Chernobyl area (Tables S3 and S4 in the Supporting Information; Figure 5). The role of NADP-ICDH in cell protection against ionizing radiation-induced oxidative damage was already suggested.43 The results of this study suggest posttranslational modification of NADP-ICDH during flax seed development in the Chernobyl area. The 2-DE spot (226/225), identified as NADP-ICDH, showed a shift in pI from 5.9 (deduced) to 6.5 (experimental) (Table S5 in the Supporting Information), possibly indicating posttranslational modification.44 The assumption of increased production of pyruvate in early stages of flax seed development in radio-contaminated area might also be supported by the abundance pattern of alcohol dehydrogen-

4.2. Updated Proteomics-Based Model for Plant Response toward Growth in Radio-Contaminated Environment

In contrast with our results with flax (Table 2), recently we showed that seed oil content decreased from 25 to 20% in soybeans harvested same year from the same fields.3 To understand molecular mechanisms of different flax and soybean response toward growth in radio-contaminated environment might help to expand our current knowledge about regulation of oil accumulation in oilseeds, which is still not well understood. On the basis of our studies, an updated model for plant adaptation to a radio-contaminated environment is proposed (Figure 6). This model is based on the data from

Figure 6. Working model of plant response toward radiocontaminated environment. This model is based on proteomic analyses of mature and developing seeds harvested from soybean and flax plants grown in radio-contaminated Chernobyl environment for two successive generations. Soybean and flax showed alterations in the abundances of proteins associated with carbon assimilation and FA metabolism. Despite significant level of soil radio-contamination, radio-nuclides transferred to soybean and flax seeds in low levels what might result in seed oil free from radio-contamination. Abbreviation: SSP, seed storage protein.

proteomic analyses of mature and developing soybean2,3 and flax4 (and this study) seeds harvested from two generations of plants grown in Chernobyl experimental fields (Figure S1 in the Supporting Information). These data support the function of SSP as not only storage reserves but also as part of seed defense45,46 and the roles of cysteine synthase and dehydrins during soybean response toward radio-contaminated environment.2,3 The results also showed that minor adjustments to multiple signaling pathways are part of flax response toward radio-contaminated environment.4 Soybean and flax showed alteration in the abundances of proteins associated with carbon assimilation and FA metabolism. Unexpectedly, seed oil content decreased in soybean3 but increased in flax (this study). Despite significant level of soil radio-contamination, radio-nuclides transferred to soybean3 and flax (Table 1) seeds in low levels might result in seed oil free from radiocontamination. However, there is still a gap in the understanding of why seed oil levels were altered in plants grown in radio-contaminated environment. Is it possible that the increased abundance of ketoacyl-[acylcarrier protein] synthase I in flax (Table S4 in the Supporting Information, Figure 5) and decreased abundance in soybean3 contribute to altered seed oil content? Is altered seed oil content the result of genetic 4804



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reflects only the authors’ views and the community is not liable for any use that might be made of information contained herein.

mutation, or is there an epigenetic or posttranslational basis? Additional studies will be needed to answer these questions.

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5. CONCLUSIONS This study is part of an ongoing investigation of seeds harvested from plants grown in the Chernobyl area. Herein we provided a proteomic view on flax seed development in the radiocontaminated Chernobyl area that resulted in an increased level of seed oil. We also presented an updated model for plant adaptation toward a radio-contaminated environment. These studies continue, and next plant generations are being analyzed using proteomics and genomic approaches to further understand the processes contributing to plant adaptation. Progress of this effort can be followed online using the dedicated webbased database available at http://www.chernobylproteomics. sav.sk that is that is accessible to the public domain.

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ABBREVIATIONS FOR ENZYMES ADH, alcohol dehydrogenase 1; AlaAT, alanine-2-oxoglutarate aminotransferase 2; ICDH, cytosolic NADP+-dependent isocitrate dehydrogenase; KAS I, 3-ketoacyl−acyl carrier protein synthase I; NADP ME, NADP-malic enzyme 1; PDC, pyruvate dehydrogenase complex; PGK, phosphoglycerate kinase; PGM, phosphoglucomutase protein; UDPGP, UDPglucose pyrophosphorylase 2

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. The location of experimental fields in the Chernobyl area. Figure S2. The 2-DE gels of proteins isolated from developing and mature flax seeds harvested from radiocontaminated and nonradioactive Chernobyl-area experimental fields. Figure S3. Reference 2-DE gel from non-radioactive Chernobyl field with locations of paired protein spots and spot IDs from control field. Table S1. The measurements of soil pH and soil particles in Chernobyl area. Table S2. Amounts of seeds used for independent protein extraction of biological triplicate analysis. Table S3. Relative volumes of 199 quantified 2-DE spots from developing seeds at 2, 4, 6 weeks after flowering and mature seeds harvested from flax grown in nonradioactive Chernobyl area. Table S4. The p values generated for 199 2-DE spot groups established for developing seeds harvested at 2, 4, and 6 weeks after flowering and maturity from non-radioactive and radio-contaminated Chernobyl experimental fields. Table S5. List of 79 proteins isolated from flax seeds grown in Chernobyl area during development identified by LC−MS/MS. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: 421-37-6943346. Fax: 421-37-7336660. E-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Seventh Framework Program of the European Union − International Reintegration Grant (MIRG-CT-2007-200165), Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Academy of Sciences (VEGA-2/0126/11), the Slovak Research and Development Agency (APVV-0740-11), and Research & Development Operational Program funded by the ERDFCentre of Excellence for White-Green Biotechnology (ITMS 26220120054). M.D. was supported by the National Scholarship Program of the Slovak Republic. We thank Prof. Ján Miernyk for English language editing and helpful critical comments. We also thank Mr. Volodymyr Sakada for technical help with maintenance of the Chernobyl fields. This paper 4805



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Radioactive Chernobyl Environment Has Produced High-Oil Flax

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