Tropospheric ozone, one of the most phytotoxic air pollutants, may specially impose in long-lived forest trees substantial reduction in productivity and biomass. European beech saplings grown in lysimeter around areas were used to monitor proteomic changes upon elevated ozone concentrations following four vegetation periods of exposure. A proteome study based on highly sensitive two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was performed to identify protein changes in European beech, the most important deciduous tree in Central Europe. Main emphasis was on identifying differentially expressed proteins after long-time period of ozone exposure under natural conditions rather than short-term responses or reactions under controlled conditions. Our results clearly demonstrate a response of European beech saplings to long-term ozone fumigation at the protein level. We indicate changes in the protein abundance of 142 protein spots; among them 59 were increased and 83 decreased following three years of elevated ozone exposure. As the first step, 40 proteins were identified by a homology driven mass spectrometric approach. Some of the identified proteins have been previously described in the context of short-term ozone responses in plants, indicating, at least for certain cellular functions, the congruence of plant reactions following short- and long-term ozone exposure. Under elevated ozone exposure, abundance of proteins related to the Calvin cycle and photosynthetic electron transport chain were decreased whereas the abundance of proteins regarding the carbon metabolism/catabolism were increased.
Tropospheric ozone, as an indirectly emitted greenhouse gas, is present in low concentrations, but minor changes in its abundance seem to have strong influences on living organisms. Mainly due to anthropogenic pollutants, such as those from biomass and fossil fuel burning, ground levels of ozone concentrations have more than doubled in the past 100 years (
Exposure to ozone has been linked to a number of altering effects in plants, including reduced growth, lower yields, accelerated senescence and necrosis in leaf tissue (
During the last years the use of proteomics in plant biology research has increased significantly (
Here we present for the first time a proteomic approach using highly sensitive two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) coupled with a homology driven mass spectrometric approach in order to identify local changes in leaf proteins from European beech saplings following three years ozone fumigation, covering two entire and two partial vegetation periods. Furthermore we used complementary transcript results performed on the same study (
The experimental design of the lysimeter and its surrounding area including the free-air ozone exposure device was previously described (
Each biological sample consisted of a pool of three leaves from one tree. Samples were ground to a fine powder using a micro dismembrator (BraunTM) without interruption of the cooling chain. Soluble proteins were extracted using 100 mg of fresh weight material according to the protocol of
Prior separation 50 µg of each sample and internal standard were labeled with 200 pmol of CyDyes diluted in N,N-dimethylformamide. A randomized sample labeling with Cy3 and Cy5 dyes was used in order to avoid systemic errors introduced during the labeling reaction. Cy2 was used to label the internal standard that consisted of equal amounts of all protein extracts from control and treatment samples within the experiment. A total of ten 2-D gels, each consisting of an internal standard, a control and a treatment sample, were used to separate proteins. For the isoelectric focusing (IEF), 24 cm and 18 cm strips were used with a linear pI gradient ranging from 4-7 and 6-11 (GE Healthcare). CyDyes labelled samples (a total of 150 µg) were mixed in rehydration solution (7 M urea, 2 M thiourea, 2 mM 2-hydroxyethyl disulfide, 2% Octyl-β-D-glucopyranoside, 0.5% Pharmalyte 3-10, 0.002% bromophenol blue and further 10% isopropanol for basic gradients) and loaded onto IPG DryStrips using in-gel sample rehydration technique for gels with pI 4-7. Proteins separated in gels pI 6-11 were cup loaded prior to IEF. After rehydration for 12 hours IEF was carried out with a maximum current setting of 50 mA/strip at 20 °C on an Ettan IPGphor Manifold (GE Healthcare). The system was programmed for strips pI 4-7 as follows: 150 V for 6 h,150-300 V for 4 h, 300-1000 V for 11.25 h, 1000-8000 V for 3 h and 8000 V for 5 h. For strips pI 6-11 following settings were used: 150 V for 5 h, 300 V for 3 h, 300-1000 V for 6 h, 1000-8000 V for 3 h and 8000 V for 1.5 h. After IEF each strip was equilibrated with 10 mL of equilibration solution (6 M urea, 75 mM Tris-HCl pH 8.8, 30% glycerol, 2% SDS, 0.002% bromophenol blue). Equilibration was performed in two steps each 15 min, with 1% w/v dithiothreitol in the first equilibration, and 2.5% w/v iodoacetamide in the second equilibration. SDS-PAGE was carried out on an Ettan DALT six system (GE Healthcare) in 12.5%, 1-mm-thick polyacrylamide gels. Electrophoretical conditions were applied as follows: 30 mA for 1 h, 48 mA for 1 h and 98 mA over night.
Gels were scanned using a Typhoon 9410 imager (GE Healthcare) at 100 μm resolution. Cy2, Cy3 and Cy5 dyes were respectively excited at 488, 532 and 633 nm.
Raw gel images were aligned with Progenesis SameSpots® software (Nonlinear Dynamics) using a master gel as reference. Virtually wrapped gels were imported in DeCyder® 6.5 software (GE Healthcare) for further 2-DE spot identification, normalization and quantification.
The low separation and labeling quality of proteins from gel 7 resulted in the exclusion of samples from tree number 7 of both groups. A total of 27 gel images (9 controls, 9 treated and 9 internal standards) were used for statistical analysis. The abundance of each protein was estimated by the volumes (sum of pixel intensity within the spot boundary). Differences in spot intensity between groups were compared using the Student´s
Preparative gels loading 700-800 µg total protein amount were run and stained with colloidal Coomassie G-250 (CBB) according to
Peptide mixtures were analyzed by on-line capillary nano HPLC (LC Packings, Amsterdam, Netherlands) coupled to a nanospray LCQ Deca XP ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Peptide digest (10 µL) were loaded onto 300 µm inner diameter x 5 mm C18 PepMap™ trap column (LC Packings, Amsterdam, Netherlands) at a flow rate of 30 µl/min. The peptides were eluted from the trap column onto an analytical 75 µm inner diameter x 15 cm C18 PepMap™ column (LC Packings, Amsterdam, Netherlands) with a 5-40% linear gradient of solvent B in 30 min (solvent A was 0.1% formic acid in 5% ACN, and solvent B was 0.1% formic acid in 80% ACN). The separation flow rate was set at 200 nl/min. The mass spectrometer operated in positive ion mode at a 1.8 kV needle voltage and a 28 V capillary voltage. Data acquisition was performed in a data-dependent mode by alternating a MS scan survey over the range
In the present study we analyzed ozone-related responses of European beech leaves following four vegetation periods of ozone fumigation under field conditions. Visible foliar symptoms represented by leaf discoloured areas were recorded for five time points on beech saplings grown inside lysimeters (n=16 trees/group - Grams, pers. comm.). Results did not show any visually symptom on foliar injury following ozone exposure for the harvesting time point of the proteomic approach (27th of July).
After image analysis of 2-D gels we could observe on average a total amount of 1350 spots in both pI ranges (pI 4-7 and pI 6-11). Our results showed that 9% of the total protein spots were differentially regulated on abundance after elevated ozone fumigation. The image analysis revealed that in the pI range 4-7, 114 spots and in the pI range 6-11, 28 spots showed different protein abundances by comparing samples fumigated with twice ambient ozone versus the control treatment (
The highest number of altered proteins according to a single metabolic pathway was observed in the Calvin cycle, where a total of seven chloroplast proteins were reduced in amount. The enzyme probable ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBisCO) activase (spot 1353) and carbonic anhydrase (spot 621), two enzymes involved in the carboxylation process by regulating the activity of RuBisCO, showed respectively decreased abundance ratios of -1.89 and -1.61 fold compared to the controls. Also the reduction part of the Calvin cycle was affected by decreased abundance levels of phosphoglycerate kinase (spot 1389). Furthermore, phosphoribulokinase (spot 1386), sedoheptulose-1.7-bisphosphatase (spot 1428), two isoforms of probable fructose-bisphosphate aldolase (spot 408/1463) and transketolase (spot 613), enzymes implicated in the regeneration of ribulose-1.5-bisphosphat, were also reduced in their amount. Similar results were observed in other plant species treated with short-term ozone exposure (
The Calvin cycle activity has been suggested to be a major sink of ATP and nicotinamide adenine dinucleotide phosphate produced during photosynthesis (
An ozone related down-regulation of enzymes associated with the Calvin cycle suggest that less triose-phosphates are produced during the CO2 fixation process, thus leading to a decreased availability of substrates for energy production (
In contrast to the Calvin cycle and the photosynthetic apparatus three proteins regarding the carbon metabolism/catabolism showed increased amounts in elevated ozone exposed beech leaves. There is strong evidence that catabolic pathways (
Further two proteins which are embedded in the mitochondrial electron transport chain, cytochrome c oxidase subunit 5B (spot 2326/2288) and ATP synthase subunit D (spot 2188), were more abundant in treated leaves. In Scots pine needles ozone led to increased enzymatic activities of cytochrome oxidase (
Although our proteome profile represents only a snap-shot of chronic ozone effects in plants grown under field conditions, it confirmed previous reported short-term ozone effects on plant species including poplar, rice and soybean. In general CO2 fixation is reduced showing decreased amounts in proteins related to the Calvin cycle. These results are accompanied by reduced amounts of photosynthetic proteins/transcripts and an overall increase in proteins involved in metabolic/catabolic pathways.
In the present work proteomic analyses, indicated complementary responses to previous reported transcript analysis from the same experiment (
We are grateful to Christophe Plomion and Stephane Claverol for very helpful comments and substantial organizational support concerning the LC-MS/MS analyses. The authors would also like to thank Sarah Sturm for excellent technical assistance and Edgar Delgado-Eckert for critical reading the manuscript. This research was carried out with financial support from the Deutsche Forschungsgemeinschaft (SFB 607) and, with respect to LC-MS/MS, by the Biodiversité Gènes et Communautés, INRA within the frame of the Network of Excellence “EVOLTREE”.
The following abbreviations have been used throughout the text:
2-D DIGE: two-dimensional fluorescence difference gel electrophoresis
ACN: acetonitrile
AOT 40: accumulated exposure over a threshold of 40 ppb
ATP: adenosine triphosphate
BSA: bovine serum albumin
CBB: colloidal Coomassie G-250
DCn: delta correlation value
ESTs: expressed sequence tags
IEF: isoelectric focusing
LC-MS/MS: liquid chromatographic- tandem mass spectrometry
MS/MS: tandem mass spectrometry
Mw: molecular weight
NaOH: Sodium hydroxide
pI: isoelectric point
RuBisCO: ribulose-1.5-bisphosphate carboxylase/oxygenase
TCA: tricarboxylic acid cycle
TRIS: tris(hydroxymethyl)-aminomethan
Schematic drawing of the experimental site. Lysimeters are represented by light blue circles. Green and red circles represent beech saplings fumigated with respectively ambient and twice ambient ozone. Numbered circles show the position of the used beech saplings in the experiment.
Schematic diagram of the experimental design in the time scale. Proteomic analyses were performed for the time point 27th July 2006.
DIGE labelled 2-D gels of separated soluble proteins from European beech leaves. Red marked protein spots indicate changes in their regulation after statistical analysis. Zoomed squares exemplify patterns of protein regulation. Spots labeled with triangles area visualized as 3-D graph in the area below.
Illustration of possible molecular changes in beech leaves exposed to elevated ozone. Red and green colored transcripts/proteins/metabolites were respectively decreased/increased in amount. Transcripts are shown in italics, whereas metabolites in bold. (CA): carbonic anhydrase; (RuBisCO activase): ribulose-1.5-bisphosphat-carboxylase/-oxygenase activase; (PGK): phosphoglycerate kinase; (PRK): phosphoribulokinase; (SBPase): sedoheptulose-1.7-bisphosphatase; (ALDP): fructose-bisphosphate aldolase; (TKTL): transketolase; (COX subunit 5B): cytochrome c oxidase subunit 5B; (Fru-6-P): fructose 6-phosphate; (Glu-6-P): glucose 6-phosphate; (ATP): Adenosine-5’-triphosphate; (ADP): adenosine diphosphate; (NADPH): reduced nicotinamide adenine dinucleotide phosphate; (NADP+): nicotinamide adenine dinucleotide phosphate (FK 1): probable fructokinase-1.
List of identified proteins by LC-MS/MS. (a) Number of protein spot on the 2-D gel; (b) Accession number from the best homologous protein in Swiss-Prot and/or TrEMBL database; (c) Q-value calculated by treatment interaction of control versus treated samples. Values were calculated based on of the Student’s
Spot number(a) | Accession number(b) | Identified protein | Q-value(c) | Average ratio(d) | Volume(e) | Standard deviation(f) | Exp.(g) | Theo.(h) | Data from the SEQUEST search | Compart.(k) | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cont | Treat | Cont | Treat | pI | Mw | pI | Mw | M(i) | Cov.(j) | ||||||
Calvin Cycle photosynthesis related proteins | |||||||||||||||
613 | Q43848 | Transketolase | 0.0054 | ↓-2.13 | 13.1 | 6.16 | 5.64 | 3.24 | 6.16 | 88 | 5.53 | 72.93 | 3 | 3.97 | chloropl. |
1389 | Q42961 | Phosphoglycerate kinase | 0.0040 | ↓-2.09 | 9.02 | 4.21 | 3.37 | 2.14 | 6.19 | 55 | 5.59 | 42.58 | 2 | 7.58 | chloropl. |
1353 | Q40281 | RuBisCO activase | 0.0024 | ↓-1.89 | 11.75 | 6.27 | 5.04 | 2.78 | 5.08 | 56 | 8.2 | 48.07 | 3 | 8.49 | chloropl. |
408 | Q9ZU52 | Probable frutctose-bisphosphate aldolase 3 | 0.0091 | ↓-1.81 | 67.51 | 36.36 | 2.96 | 1.56 | 7.12 | 40 | 6.08 | 38.08 | 4 | 10.75 | chloropl. |
621 | P27141 | Carbonic anhydrase | 0.0043 | ↓-1.61 | 215.32 | 154.59 | 6.87 | 4.88 | 6.79 | 28 | 6.19 | 23.96 | 1 | 3.6 | chloropl. |
1463 | P16096 | Fructose-bisphosphate aldolase | 0.0001 | ↓-1.55 | 27.49 | 17.6 | 7.65 | 4.5 | 6.51 | 53 | 5.8 | 37.7 | 4 | 7.19 | chloropl. |
1428 | P46283 | Sedoheptulose-1.7 bisphosphatase | 0.0077 | ↓-1.55 | 12.27 | 7.72 | 3.49 | 1.67 | 5.16 | 53 | 6.17 | 42.41 | 3 | 10.86 | chloropl. |
1386 | P27774 | Phosphoribulokinase | 0.0021 | ↓-1.53 | 29.72 | 18.74 | 13.99 | 8.43 | 5.33 | 55 | 5.22 | 39.18 | 5 | 10.7 | chloropl. |
490 | Q9ZUC1 | Quinone oxidoreductase-like protein Atlg23740 | 0.0012 | ↓-1.64 | 181.16 | 113.29 | 6.12 | 5.39 | 6.49 | 35 | 8.46 | 40.98 | 7 | 22.4 | chloropl. |
Carbon metabolism/catabolism | |||||||||||||||
1563 | Q9SIDO | Probable fructokinase-1 | 0.0075 | ↑ 1.32 | 1.04 | 1.38 | 0.39 | 0.22 | 5.17 | 49 | 5.31 | 35.28 | 2 | 4.8 | pl. memb. |
2326 | P80499 | Cytochrome c oxidase subunit 5B | 0.0011 | ↑ 1.7 | 1.72 | 2.93 | 0.29 | 0.74 | 4.88 | 18 | 4.96 | 3.1 | 3 | 6.13 | mitoch. |
2288 | P80499 | Cytochrome c oxidase subunit 5B | 0.0006 | ↑ 5.19 | 0.64 | 3.47 | 0.47 | 1.92 | 4.87 | 20 | 4.96 | 3.1 | 1 | 3.68 | mitoch. |
2188 | Q9FT52 | ATP synthase subunit d | 0.0040 | ↑ 1.77 | 1.39 | 2.4 | - | 0.66 | 5.46 | 26 | 5.09 | 19.45 | 2 | 3.8 | mitoch. |
List of peptide sequences of proteins identified by LC-MS/MS.