Proteomic Analysis of Multiprotein Complexes in the Thylakoid

Proteomic Analysis of Multiprotein Complexes in the Thylakoid Membrane upon Cadmium Treatment Marco Fagioni, Gian Maria D’Amici, Anna Maria Timperio, and Lello Zolla* Department of Environmental Sciences, Tuscia University, Viterbo, Italy Received July 8, 2008

The time course of the thylakoid membranes proteomic profile changes upon cadmium (Cd) addition to hydroponic Spinacia oleracea L. plants has been investigated. Two different proteomic approaches have been used: blue native gel electrophoresis followed by SDS-PAGE (2D BN-SDS-PAGE) and sucrose density gradient ultracentrifugation followed by RP-HPLC. Chlorophyll (Chl) and xanthophylls concentrations, together with ESR and real time PCR measurements, were also performed to get a complete overview of all photosystem changes. Cd only accumulated in basal leaves, that therefore were prevalently investigated for assessment of Cd induced changes. Here, Cd strongly reduced Chl concentration, especially Chl a. During the first 15 days of treatment, native electrophoresis system revealed high sensitivity of PSI to Cd, while minor effects on PSII were observed. Cytochrome b6/f and the ATP-synthase complex did not change following the Cd treatment. A significant reduction of antenna proteins of PSI was observed, while PSII antennae were affected to a minor extent, with exception of the isomeric Lhcb1.1 which decreased significantly already at the onset of the treatment. Some PSII core proteins were overexpressed, but showed reduced activity. No new protein was formed and no specific protein disappeared in the photosynthetic apparatus of Cd-treated leaves. Upon removal of Cd, a rapid resynthesis of total Chl and a significant resynthesis of Lhcb1.1 antenna were observed, suggesting that Cd affects specifically the photosynthetic apparatus of spinach basal leaves, replacing other metal ions inside proteins. Keywords: Cadmium treatment • thylakoid membrane • blue native PAGE • spinach

Introduction Cadmium (Cd), a divalent ion with density ) 8.6 g cm-3, is a heavy metal widespread in areas with high levels of anthropogenic pollution.1 Cd is released into the environment by power stations, heating systems, metal-working industries, waste incinerators, urban traffic, cement factories, and as a byproduct of phosphate fertilizers. Remarkably, Cd is easily transferred from the environment into the food chain.2 Cd has negative effects on plant biology. Although it has not been clearly established how plants respond to heavy metals, it is well-known that Cd exerts its phytotoxicity by interfering with several basic events of plant growth, development and physiology, among which are mineral nutrition, water balance and gas-exchange, membrane function, cell metabolism and defense against oxidative stress.3 Cd is readily taken up by roots and translocated into aerial organs where it can accumulate to high levels.4 Cd uptake is species-specific5 as well as its distribution into plant organs.1 Plant responses to Cd2+ exposure, in fact, are known to influence Cd accumulation rates; enhanced intracellular binding, for instance, drives the accumulation of Cd.6 In cucumber, Cd was found localized * To whom correspondence should be addressed. Prof. Dr. Lello Zolla, Department of Environmental Sciences, Universita` “La Tuscia” Largo Dell’Universita`, 01100 Viterbo, Italy. Tel.: +39-0761-357100. Fax: +39-0761357179. E-mail: [email protected].

310 Journal of Proteome Research 2009, 8, 310–326 Published on Web 11/26/2008

prevalently in the 4-5 basal leaves.7 Regarding Cd effects, Cd stimulates the production of metabolic enzymes in the roots,8 while in leaves, the most evident effect of Cd toxicity is chlorosis. This symptom seems to develop because Cd triggers the inhibition of root Fe(III) reductase, which leads to Fe(II) deficiency in leaves, and to associated reduction of photosynthesis.9 Because of the chemical similarity, Cd can replace Zn, Fe and Ca into the prosthetic group of many proteins. Cd may replace Zn ions in different metal-containing enzymes, such as carbonic anhydrase in Cyanobacteria10 and DNA-binding proteins (Zn-fingers) in eukaryotic cells.11 Some metal ions, such as Fe2+ in [2Fe-2S] clusters12 or Ca2+ in concanavaline A13 can be substituted by Cd.14 Cd can also inactivate same important enzymes by Cd-binding sulfydryl-groups (-SH).2 It has also been hypothesized that Cd may replace the central Mg ion in the chlorophyll (Chl) molecules,11 and may interfere directly with enzymes of the Chl biosynthesis pathway or with the correct assembly of the pigment-protein complexes of the photosystems.4 Finally, Cd may also affect cellular processes, altering membrane permeability due to changes in lipid composition and/or lipid peroxidation and interacting with nucleic acids.15 In fact, Cd is also supposed to directly or indirectly cause the formation of reactive oxygen species (ROS) which alter redox status,16 and are responsible for significant Chl loss, damages to membranes.17 10.1021/pr800507x CCC: $40.75

 2009 American Chemical Society

Hydroponic Spinach Plants under Cadmium Treatment A main effect of this metal, observed in most plants studied to date, is the inhibition of photosynthesis. However, controversial results have been obtained. Several studies demonstrated that the light reactions of photosynthesis are not sensitive to Cd, while stomatal movement and Calvin cycle enzymes are primarily affected.18,19 In other works, however, Cd was shown to inhibit the photochemical activity of choloroplasts, particularly photosystem II (PSII).20 Cd also markedly reduces ferredoxin (Fd)-dependent NADP+ photoreduction, which indicates that the metal interferes with electron transport on the reducing side of photosystem I (PSI).21 In Cucumis sativus L., Cd specifically reduced photosystem I complexes.7 However, Cd effects on both photosystem are not the same in all plants.22 Recently, Kieffer et al. analyzed by 2D DIGE the proteome of the young leaves exposed to 20 µM Cd during 14 days.23 They were able to demonstrate a deleterious effect on primary carbon metabolism and oxidative stress response mechanism. In the present study, the time course of changes induced by Cd stress in the proteomic profile of thylakoid membranes of the higher plant Spinacia oleracea L. was characterized. To get a complete overview of all photosystem changes, two complementary proteomics approaches were used: two-dimensional blue native sodium dodecyl sulfate gel electrophoresis (2D BNSDS-PAGE) and sucrose density gradient ultracentrifugation followed by reversed-phase high-performance liquid chromatography (RP-HPLC). The use of nondenaturing conditions provides protein complexes in native form, allowing a better analysis of the physiologic state of the organism. On the other side, proteomic analysis of light-harvesting antenna proteins, the most affected proteins in both photosystems, was performed by using sucrose density gradient ultracentrifugation followed by RP-HPLC. The investigation was carried out on basal leaves, after verification that Cd prevalently accumulates on these leaves.

Material and Methods Plant Material and Experimental Treatments. Spinach seeds (S oleracea L.) were disinfected in 1% (v/v) NaClO solution for 10 min followed by thorough washing in deionized water. Seeds were then transferred to a beaker containing 30% (w/v) PEG 8000 for 72 h. Then seeds were washed again in 1% (v/v) NaClO solution and soaked in deionized water for 5 h to rehydrate, with continuous aeration. The seeds were then left to germinate on moist filter paper for 2-3 days. Half of the germinated seeds were sown in moist vermiculite in halfstrength Hoagland24 and grown for about 5 days (four-leaf stage). After the initial growth period in the seedbed, seedlings were carefully removed from the seedbed, and were thoroughly washed free of any adhering particles under tap water. The spinach plantlets were then grown hydroponically in a growth room under a 14-h light period at a light intensity of 260-350 µmol m-2 s-1 and the temperature was set at 24/20 °C (day/ night). The seedlings were transferred to 0.9 dm3 PVC buckets. When the plants were 20 days old, half of the seedlings were grown in half-strength Hoagland nutrient solution with Cd at different concentration (20, 50, 100 µM) under continuous aeration at pH 5.5,25 while other half were grown without Cd (control plants). Nutrient solution was changed every 3 days in both normal and Cd-treated plants. It is important to remind that Hoagland solution contains 50 mM K2SO4, 0.1 M MgSO4 · 7H2O, 1 mM CaSO4 · 2H2O, 8 mM NaH2PO4, 0.1 KNO3

research articles (macronutrients), 0.014 mM MnCl2 · 4H2O, 0.76 µM ZnSO4 · 7H2O, 0.32 µM CuSO4 · 5H2O and 0.08 µM H2MoO4 (micronutrients). Basal and apical leaves were harvested separately from plants after 0, 5, 10, 15, 30, and 33 days of Cd treatment. At the same days, basal and apical leaves were harvested also from the control plants. The same age controls plant leaves were used as control in all the experiments performed. Regarding the leaves investigated, it is important to underline that 20 day spinach plants (before the beginning of Cd treatment) had six leaves. The first four basal leaves (1-2 and 3-4) were analyzed separately from the two apical ones (5-6) for their biochemical composition and structure. All the leaves were frozen in liquid nitrogen before using them for biochemical measurements. In the reconstitution experiment, plants growth for 30 days in the presence of Cd were divided in three culture lines: one was maintained in the presence of Cd as a control, one had Cd removed, and the last one had the Cd substituted by 7.65 µM Zinc sulfate eptahydrate (10-fold with respect to the Zn2+ amount of that present in the Hoagland solution) was added. After 3 days, basal leaves from three plant coltures were harvested and frozen in liquid nitrogen. Isolation of Intact Chloroplasts. Fully developed spinach leaves were used to isolate intact chloroplasts using the method of Nishimura et al. with minor modifications.26 Protoplasts were prepared from freshly harvested spinach leaves following the method reported previously.26 Broken protoplasts (0.5 mL) were directly layered on top of a 15 mL linear sucrose gradient (35-60%, w/w), dissolved in 0.02 M Tricine-NaOH buffer (pH 7.5), and centrifuged at 50 000g for 3 h at 4 °C using a BeckmanSpinco SW 25-3 rotor. In both control and Cd-treated spinach plants (both apical and basal leaves), chloroplast numbers were determined as described by Dudley.27 Isolation of Thylakoid Membranes. For the separation of the thylakoid membrane of chloroplasts from both control and Cd-treated spinach plants, leaves were ground to a powder in liquid nitrogen and subsequently homogenized in an ice-cold 20 mM Tricine, pH 7.8, buffer, containing 0.3 M sucrose and 5.0 mM magnesium chloride (B1 buffer). The homogenization was followed by filtration through one layer of Miracloth (Calbiochem, San Diego, CA) and centrifugation at 4500g for 10 min at 4 °C. The pellet was suspended in B1 buffer and centrifuged again, as above. This second pellet was resuspended in 20 mM Tricine, pH 7.8, buffer, containing 70 mM sucrose and 5.0 mM magnesium chloride (B2 buffer) and centrifuged at 4500g for 10 min. A third pellet was then collected which contained the thylakoid membrane. 2D BN-PAGE-SDS PAGE in Basal and Apical Leaves. 1D BNPAGE of integral thylakoid proteins was performed according to Ku ¨ gler et al.28 and Suorsa et al.29 with the following modifications. Thylakoid membranes were washed with washing buffer (330 mM sorbitol, 50 mM BisTris-HCl, pH 7.0), and 250 mg/mL Pefabloc as a protease inhibitor (Roche, Indianapolis, IN), collected by centrifugation (3500g for 2min at 4 °C), and resuspended in 25BTH20G (20% (w/v) glycerol, 25 mM BisTris-HCl, pH 7.0, and 250 mg/mL Pefabloc). An equal volume of resuspension buffer containing 2% (w/v) n-dodecylβ-D-maltoside (DDM; Sigma, St. Louis, MO) was added under continuous mixing, and the solubilization of membrane protein complexes was allowed to occur for 3 min on ice. Insoluble material was removed by centrifugation at 18 000g for 15 min. The supernatant was mixed with 0.1 vol of Coomassie Blue Journal of Proteome Research • Vol. 8, No. 1, 2009 311

research articles solution (5% (w/v) Serva blue G, 100 mM BisTris-HCl, pH 7.0, 30% (w/v) sucrose, and 500 mM ε-amino-n-caproic acid) and loaded onto a 0.75 mm thick 5-12.5% (w/v) acrylamide gradient gel (180 × 160 mm). For each sample, the same amount of protein (100 µg) was loaded. Protein concentration was estimated by the DC protein assay (Bio-Rad, Hercules, CA) before the addition of Coomassie Blue solution. Electrophoresis run was performed at 4 °C. We used a Protean II xi cell electrophoresis system (Bio-Rad) for the first dimension applying a constant voltage of 90 V overnight and gradually increasing this value up to 250 V the next day until the run was complete. For separation of proteins in the second denaturing dimension, the lanes of the 1D BN gel were excised and incubated for 30 min in SDS sample buffer containing 5% (v/v) β-mercaptoethanol and 6 M urea for 30 min at room temperature. The lanes were then layered onto 1 mm thick SDS-PAGE gels30 with 15% (w/v) acrylamide and 6 M urea in the separating gel. Proteins were separated overnight at constant current 10 mA per gel and 13 °C using a Protean II xi cell electrophoresis system (180 × 160 mm, 1 mm thick). The proteins were stained using Blue silver.31 Image Analysis. First dimension gel images were digitized using a ImageScanner II (GE Healthcare, Uppsala, Sweden) with a resolution of 300 dpi and 16-bit greyscale pixel depth. Image analysis was carried out with TotalLab software vers. 2.01 (Nonlinear Dynamics, Newcastle, England), which allows bands detection, quantification, and background subtraction. Each stage of leaves development (both of control and Cd-treated plants) was analyzed in 5 replicates. For each replicate, leaves were collected from different plant cultures. Moreover, into the same culture, leaves from three different plants (three leaves for plant) were pooled, in agreement to Karp et al.32 1D BNPAGE protein bands were selected for profile analysis only if they were found and positively assigned in all the 5 replicates of each sample. To compensate for subtle difference in sample loading, the volume of each band was normalized as relative volume. This normalization method divides each spot volume value by the sum of total band volume values to obtain individual relative band volumes. Total band volume refers to the sum volume of all bands chosen for analysis. The normalized volume of each bands was plotted using the software SigmaPlot vers. 9.0 (Systat, Erkrath, Germany). Second dimension gel images were digitized using a ImageScanner II with a resolution of 300 dpi and 16-bit gray scale pixel depth. Images were analyzed with the Progenesis SameSpots software vers. 2.0 (Nonlinear Dynamics). A match set was created from the protein patterns of five replicate gels for each independent chloroplast extract (at day ) 0 and 10 of Cd treatment) which allows spot matching among multiple gels. Spot quantities of all gels were normalized to remove nonexpression-related variations in spot intensity. To normalize the raw spot volume (evaluated in terms of optical density), each spot on gel image was expressed relative to the total volume of all spots on that image which are matched and presented in all the gels of the experiment. Statistical analysis separated proteins that significantly increased or decreased after the treatments. A Student’s t test was performed in order to compare the two groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0.05. Image analysis allowed the identification of 18 proteins differently expressed by more than 50% (p < 0.05). 312

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Fagioni et al. In-Gel Digestion. Bands and spots, respectively, from 1D and 2D gels were carefully excised and subjected to in-gel trypsin digestion according to Shevchenko et al. with minor modifications.33 The gel pieces were swollen in a digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/µL trypsin (modified porcine trypsin, sequencing grade, Promega, Madison, WI) in an ice bath. After 30 min, the supernatant was removed and discarded, 20 µL of 50 mM NH4HCO3 was added to the gel pieces, and digestion was allowed to proceed at 37 °C overnight. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Prior to mass spectrometric analysis, the peptide mixtures were redissolved in 10 µL of 5% FA (formic acid). Peptide Sequencing by Nano-RP-HPLC-ESI-MS/MS. Peptide mixtures were separated using a nano flow-HPLC system (Ultimate; Switchos; Famos; LC Packings, Amsterdam, The Netherlands). A sample volume of 10 µL was loaded by the autosampler onto a homemade 2 cm fused silica precolumn (75 µm i.d.; 375 µm o.d.; Reprosil C18-AQ, 3 µm, AmmerbuchEntringen, Germany) at a flow rate of 2 µL/min. Sequential elution of peptides was accomplished using a flow rate of 200 nL/min and a linear gradient from Solution A (2% acetonitrile; 0.1% formic acid) to 50% of Solution B (98% acetonitrile; 0.1% formic acid) in 40 min over the precolumn in-line with a homemade 10-15 cm resolving column (75 µm i.d.; 375 µm o.d.; Reprosil C18-AQ, 3 µm, Ammerbuch-Entringen). Peptides were eluted directly into a high capacity ion trap (model HCTplus Bruker-Daltonik, Germany). Capillary voltage was 1.5-2 kV and a dry gas flow rate of 10 L/min was used with a temperature of 200 °C. The scan range used was from 300 to 1800 m/z. Protein identification was performed by searching in the National Center for Biotechnology Information nonredundant (NCBInr) database using the MASCOT program (http://www.matrixscience.com). The following parameters were adopted for database searches: complete carbamidomethylation of cysteines, partial oxidation of methionines, peptide mass tolerance (1.2 Da, fragment mass tolerance (0.9 Da and missed cleavages 2. For positive identification, the score of the result of [-10 × Log(P)] had to be over the significance threshold level (P, 0.05). Separation of PSI and PSII Membrane Proteins by Sucrose-Gradient Ultracentrifugation and Analysis of Contents by HPLC. This technique allows the separation of the antennae proteins of PSI and PSII into distinct chromatographic peaks34,35 and makes it possible to analyze them quantitatively. Purification of thylakoid membrane multiprotein complexes (MPCs) was performed according to the method of Croce et al.36 Briefly, the thylakoid membrane was solubilized by DDM to a final concentration of 1%. After stirring for 10 min at 4 °C, the sample was centrifuged for 10 min at 20 000g, and 1 mL aliquots of the supernatant were loaded onto 0.1-1 M sucrose gradients, containing 5 mM Tricine, pH 7.8, and 0.03% (w/v) DDM. After centrifugation for 24 h at 111 000g in a Model SW41 rotor (Beckman) at 4 °C, five green bands were distinguishable36 that were suitable for direct investigation by RP-HPLC-ESI-MS. To optimally resolve the protein components of the antenna systems of PSII and PSI as isolated complexes by sucrose gradient, we used an analytical (250 × 4.6 mm i.d.) size column, packed with the same 5 mm spherical Vydac C-4 stationary phase. The separation of the protein components of the PSII and PSI antenna system was obtained by the following proce-

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Hydroponic Spinach Plants under Cadmium Treatment dure. The Vydac C-4 column was pre-equilibrated with 38% (v/v) aqueous acetonitrile solution containing 0.05% (v/v) TFA, and samples were eluted from a column with a gradient of 40-65% acetonitrile in 45 min. The flow rate was 1.0 mL/min with the analytical column. The HPLC-ESI-MS experiments were performed by an ion trap Esquire 3000 plus (Bruker Daltonik, Bremen, Germany). Details of instrumental setup and tuning are given in Zolla et al.35 The 1 mL/min flow through the analytical column was split postcolumn, with 50 µL/min entering the mass spectrometer, and 950 µL/min going to the UV and fluorescence detector. For analysis with pneumatically assisted ESI, an electrospray voltage of 3-4 kV and a nitrogen sheath gas flow were employed. The temperature of the heated capillary was set to 300 °C. Protein mass spectra were recorded by scanning the first quadrupole, the scan range being 500-2000 amu. Pigment Determination by High Performance Liquid Chromatography. Pigments were extracted from samples by grinding the leaves in liquid nitrogen with degassed 100% acetone at 0-4 °C. On occasion, a small amount of NaHCO3 (0.1 g g-1 plant material) was added to the plant material before extraction to ensure no acidification of the extract occurred; however, this had no influence on carotenoid composition. The pigment extracts were filtered through a 0.2-mm membrane filter and applied immediately to a HPLC column. Pigment composition was determined by HPLC by using a Waters NovaPak C18 radial compression column according to Johnson et al.37 Additionally, a Spherisorb ODS-1 column (5-mm particle size, 250 × 4.6 mm i.d.) was used based on the method described by Gilmore and Yamamoto.38 To calibrate, the method pigments were first isolated by HPLC. Then, isolated fractions were dried under nitrogen and dissolved in the methanol, and pigment concentrations were determined using the coefficients of extinction indicated in Val et al.39 The method was calibrated by injecting known amounts of pure pigments, and plotting the peak area (integrator counts) versus quantity of pigment injected. The curve line was made using increasing concentrations of each pure pigment and quantifying each concentration spectrophotometrically. Data are presented as milligrams per 100 g of basal leaf dry weight.40 Statistical Analyses. Chl and pigment concentration values of leaves were subjected to analysis of variance (ANOVA) for treatment effects at each time point. If the analysis determined significant differences among means, the analysis of variance was rerun and included both Tukey’s and Scheffe’s mean separation tests (R ) 0.05) for distinguishing among significantly deviating means. Cd Ion Determination by Atom Absorption. Metal contents were determined after a wet digestion with HNO3:H2O 1:1 (v/ v) by inductively coupled plasma atomic emission spectrometry (ICP-AES).41 ESR Spectroscopy. ESR spectra and kinetics were recorded at room temperature with a Bruker ESP300 spectrometer equipped with a TE110-mode resonator, using 20-mW power at 9.6 GHz microwave frequency. A sample volume of approximately 50 µL was drawn into glass capillaries, sealed and measured directly under continuous, strong white light illumination. Quantitative mRNA Expression Analysis. Total RNA from spinach leaves was extracted from 50 mg of leaf tissue using the RNeasy Plant Mini Kit (Qiagen, CA). For each sample, the extraction was repeated three times (3 biological replicates). The samples were treated with DNA-free kit (Ambion) to

remove any contamination from DNA. After this step, we checked the purity of RNA by PCR without retrotrascription using LHC1.1 gene specific primers. Quantitative polymerase chain reactions (qPCRs) were performed with the Quantitect SYBR Green RT-PCR kit (Qiagen, CA) with a one-step reaction. qPCR reactions were assembled in a total volume of 25 µL as described by the protocol of the kit with some modifications: 12.5 µL of the 2× QuantiTect SYBR Green RT-PCR Master mix, 0.25 µL of the Quantitect RT mix, the specific primers for Lhcb1.1, Lhcb1.2, Lhcb1.3 gene (LHC1.1Fw, 5′-GGTAAGGCGGTTAAGCTTAA-3′; LHC1.1-Rw, 5′-CACCAGTAAGGTAGGATGGA-3′; LHC1.2-Fw, 5′-CTCCCTTGCTGGAAAGGCCA-3′; LHC1.2 Rw, 5′-AACCATAGTCACCGGGGAAT3′; LHC1.3-Fw, 5′-GGAAAGCCCAAAACTGTTCA-3′; LHC1.3 Rw, 5′-CCACCTGCAGTGGATAACT-3′) at 0.2 µM final concentration, 1 µL of RNA and MQ water to 25 µL. qPCR reactions were always carried out in triplicate wells, using the following conditions: 30 min at 50 °C for the reverse transcription reaction, 15 min at 95 °C to inactivate the reverse trascriptase polymerase, followed by a total of 40 three-temperature cycles (15 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C), and 10 s at 50 °C. The gene expression was normalized to 18S rRNA gene of spinach (18S-Fw, 5′-TTTTAGGCCACGGCCGTTTG-3′; 18S-Bw, 5′-GTACAAAGGGCAGGGACGTA-3′). To quantify the qPCR amplification results, cDNA plasmid standards, specific for Lhcb1.1 gene and 18S rRNA, were constructed. Specific amplifications could not be detected at the conditions used in the Real-Time assays (not shown). To construct the cDNA standards, total RNA from spinach leaves was extracted, and the light harvesting complex gene Lhcb1.1 and 18S cDNA fragments were synthesized by RT-PCR, using the same primers previously described. Each of these fragments was purified with QIAquick PCR Purification Kit (Qiagen), cloned into pGEM-T Easy Vector System (Promega, Madison, WI), and transformed into DH5R competent cells. Plasmid DNA was purified from transformed cells using QIAGEN Plasmid Mini Kit (Qiagen). The cDNA plasmid concentration was measured by NDR ND1000 Nanodrop (Nanodrop Inc., Wilmington, DE). The corresponding copy number was calculated using the following equation: 1 µg of 1000 bp DNA ) 9.1 × 1011 molecules. In each qPCR amplification, serial dilution (10-fold) of plasmid cDNA, ranging from 10-1 to 10-7 input cDNA copies was used as a standard curve (each standard curve was performed as triplicate). All qPCR data were elaborated by the MxPro v3.2 software (Stratagene).

Results Cd Accumulation and Pigment Analysis. To 20 day spinach plants, growing in hydroponic culture with Hoagland nutrient solution, Cd was added. Differently to other species,23 spinach plants did not showed significant changes at lower Cd concentration (20, 50 µM); therefore, we decided to perform the investigation at 100 µM Cd. The first four basal leaves (1-2 and 3-4) were analyzed separately from the two apical ones (5-6) for their biochemical composition and structure. In fact, as expected,7 after 10 days of Cd treatment, no Cd was detected in apical leaves, while in the basal leaves, the Cd concentration ranged between 7.7 ( 0.1 (the first two basal leaves) and 3.9 ( 0.1 (the second ones) nmol cm-2. Since Cd was localized in the four basal leaves, we prevalently focused on these leaves for further analyses. Two new small leaves (7-8) appeared after 20 days of Cd treatment, but they were not used for our investigation. Journal of Proteome Research • Vol. 8, No. 1, 2009 313

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Figure 2. Time course of leaf photosynthetic pigment concentrations determined by HPLC analysis on Cd-treated spinach plants: Violaxanthin, Neoxanthin, Lutein. Data are presented as milligrams per 100 g of leave’s dry weight. *** (P < 0.001) statistically different from day 0 value. One-way ANOVA test.

Figure 1. (A) Time course of Chl concentrations measured in the thylakoid membranes of basal and apical spinach leaves treated with Cd. The total Chl concentration was estimated by spectrophometer at 652 nm by using solutions with the same number of chloroplasts. * (P < 0.05), ** (P < 0.01), *** (P < 0.001) statistically different from same day basal leaves value. The dotted line shows the total Chl concentration determined after 3 days of Cd removal, both in leaves that were simply fed Hoagland media plus Cd solution (dash-dot line) or Hoagland media supplemented with zinc (dotted line). +++ (P < 0.001, referred to Cd free value) statistically different from 33 day basal leaves value. ### (P < 0.001, referred to Zn value) statistically different from 33 day basal leaves value, One-way ANOVA test followed by both Tukey’s and Scheffe’s means comparison test was performed. (B) Time course of Chl a and Chl b concentrations measured in the thylakoid membranes of basal spinach leaves treated with Cd. * (P < 0.05), ** (P < 0.01), *** (P < 0.001) significantly different from day 0 Chl a and Chl b value. Oneway ANOVA followed by both Tukey’s and Scheffe’s test.

The Cd-treated basal leaves showed a reduction in the total number of chloroplasts (from 1.42 ( 0.16 to 1.022 ( 0.21 number of chloroplast per 100 µm2 cell area) with respect to controls plants. Spectrophotometric measurements showed that the total Chl concentration ([Chl]tot) of apical leaves (Figure 1A) changed similarly to the control leaves (supplementary Figure 1 in Supporting Information), indicating that photosynthetic apparatus of apical leaves is not affected by Cd. On the contrary, [Chl]tot of basal leaves decreased constantly throughout the Cd treatment. In particular, Chl a ([Chl]a) decreasing significantly more than [Chl]b, especially during the first 10 days of treat314


ment (Figure 1B). Regarding xanthophyll pigments, in basal leaves, some xanthophylls (lutein, neoxanthin and violaxanthin) increased throughout the Cd treatment (Figure 2). The effect was particularly evident for lutein in leaves treated with Cd for 15 days. Contrary to what was observed in spinach plants, grown in iron deficiency at 300 µE m-2 s-1,42 no formation of zeaxanthin and antheraxanthin was recorded throughout the Cd treatment, supporting that the chlorosis observed in the presence of Cd may be not only attributed to the inhibition of root Fe uptake, which leads to Fe(II) deficiency in leaves,9 but other mechanisms are involved. To perform recovery experiments, Cd treatment was stopped in some plants after 30 days of stress, whereas in other plants, Cd was replaced with 7.65 µM Zn ions (i.e., 10 times more than the amount present into the Hoagland solution). We have chosen this extreme exposure time with the aim to check if recovery is present also in plants showing a visible damage. Only the basal leaves (3-4) were examined because most of 1-2 leaves were dried. A resynthesis of the total Chl was recorded just after 3 days of the reconstitution experiment in both plants after 30 days of stress. The presence of Zn ions did not further increment resynthesis (see Figure 1A dotted line). Similarly, upon Cd removal, lutein content decreased again, although its total amount remained higher than in control plants (Figure 2). Proteomic Analysis. BN-PAGE is the preferred electrophoretic system for the analysis of hydrophobic membrane proteomes as it does not seem to suffer from the problems encountered in 2D electrophoresis with IEF.43,44 These high resolution 2D gel systems aim at identifying MPCs with respect to their subunit composition. However, in the case of lightharvesting antenna proteins, for which 2D BN-SDS-PAGE showed same resolution limits,43 proteomic analysis was performed by using sucrose density gradient ultracentrifugation followed by RP-HPLC (see later). BN-PAGE was performed with thylakoids isolated from both unstressed (control) basal leaves and Cd stress basal leaves after 0, 5, 10 and 15 days of treatment. 1D BN-PAGE. BN-PAGE was performed with thylakoids extracted from apical and basal leaves of both unstressed

Hydroponic Spinach Plants under Cadmium Treatment

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Figure 3. Blue Native PAGE profile of thylakoid membrane protein complexes (basal leaves after 0, 5, 10 and 15 days upon 100 µM Cd treatment). Each strip of panel (A) was loaded with the same amount of protein (100 µg), as estimated by the DC protein assay (BioRad). In panel (B), to compare the complex abundance during the development of Cd stress, a semiquantitative analysis was obtained quantifying the volume of each band with the image analysis software TotalLab vers. 2.01 (Nonlinear Dynamics). To minimize the effect of technical variation, the normalized volume data of each band reported refer to the average of five replicates. One-way ANOVA test. * (P < 0.05), ** (P < 0.01), *** (P < 0.001) statistically different from day 0 value.

(control) and Cd stress plants, after 0, 5, 10 and 15 days of treatment. Each stage of leaves development (both of control and Cd-treated plants) was analyzed in 5 replicates. It is important to remark that BN-PAGE performed on both apical and basal leaves of control plants did not show significant differences over a period of 15 days (data not show), indicating that photosynthetic complexes did not change over the leaf aging corresponding to the experimental period. Figure 3A compares the BN gel profile of Cd-treated thylakoid MPCs, extracted from basal leaves after 0, 5, 10 and 15 days of treatment. The photosynthetic complexes contained in the gel bands were identified by in-gel trypsin digestion and by tandem mass spectrometry. As reported previously, a limited number of peptides was obtained upon trypsin digestion of these highly hydrophobic proteins, but hardly sufficient for unequivocal MPCs identification.45 All protein complexes identified with this procedure are listed in Table 1. Interestingly, most complexes identified by mass spectrometry analysis corresponded to those previously identified by the apparent molecular masses of the protein complexes43,44 but with the exception that the supercomplexes at 709 kDa appear to be a PSI multimeric form rather than a complex containing both PSI and PSII proteins.43,44 All these identifications were also confirmed by second dimension analysis (see below).

To compare the abundance of the protein complexes during the development of the Cd stress, a semiquantitative analysis of each band was performed with the image analysis software TotalLab vers. 2.01 (Nonlinear Dynamics) (Figure 3B). For each sample, the same amount of protein (100 µg), measured after the solubilization step, was loaded. To minimize the effect of technical variations, semiquantitative analysis reported in Figure 3B refers to the average of five replicates.42 Statistical significance was evaluated by one-way analysis of variance (ANOVA, p < 0.05). The PSI supercomplexes with the apparent molecular mass of 709 kDa showed a reduction of the band volume with progressing Cd stress. These supercomplexes disappeared completely after 15 days of Cd treatment, indicating that the super organization of thylakoid membranes was strongly compromised and that PSI complex was strongly damaged. The green large band at 580 kDa, where the entire PSI (RCI + LHCI), the PSII core dimer, ATPase (ATP-synthase) and Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) comigrated, also showed a significant decrease of volume after 15 days of Cd treatment. Since both mass spectrometry analysis and second electrophoretic dimension (see below) confirmed that ATPase remained constant during the development of Cd stress, it can be inferred that the observed decrease in the 580 kDa band density is due to a reduction of PSI (RCI Journal of Proteome Research • Vol. 8, No. 1, 2009 315

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Fagioni et al. a

Table 1. Proteins Identified in 1D BN-PAGE of Spinach Thylakoid

peptides identified by MS/MS subunit name

m/z

Mascot score%

charge state

start-end

sequence

Band at 709 kDa: PSI Supercomplex 3+ 70-94 GFTPPELDPNTPSPIFAGSTGGLLR 3+ 95-110 KAQVEEFYVITWESPK 2+ 96-110 AQVEEFYVITWESPK 2+ 111-125 EQIFEMPTGGAAIMR 2+ 126-132 EGPNLLK 2+ 36-145 KEQCLALGTR 2+ 159-171 VFPSGEVQYLHPK 2+ 184-191 QGVGLNMR 2+ 204-212 FTGKQPYDL

NCBI accession number

Photosystem I reaction center subunit II, chloroplast precursor (Photosystem I 20 kDa subunit) (PSI-D) [Spinacia oleracea]

847.60 652.14 913.51 826.02 385.87 588.47 750.99 437.89 534.96

56 28 71 58 28 51 52 63 28

Photosystem I P700 chlorophyll a apoprotein A2 [Spinacia oleracea]

660.45 493.09 425.79 838.97 693.97 716.54 737.93 449.54

56 35 34 79 38 52 58 53

2+ 3+ 2+ 2+ 3+ 3+ 2+ 2+

8-19 8-20 303-314 397-410 452-469 470-490 552-564 685-692

FSQGLAQDPTTR FSQGLAQDPTTRR DLLEAHIPPGGR DYNPEQNEDNVLAR QILIEPIFAQWIQSAHGK TSYGFDVLLSSTSGPAFNAGR DFGYSFPCDGPGR TPLANLIR

gi|11497524

Chain F, The Structure Of A Plant Photosystem I Supercomplex At 3.4 Angstrom Resolution [Spinacia oleracea]

487.91 674.55 450.45 719.82 843.59 563.03 577.1

52 71 40 55 76 34 39

2+ 2+ 2+ 3+ 2+ 2+ 2+

1-9 31-43 52-58 59-78 118-132 133-142 143-154

DIAGLTPCK LYADDSAPALAIK RFDNYGK YGLLCGSDGLPHLIVSGDQR EIIIDVPLASSLLFR GFSWPVAAYR ELLNGELVDNNF

gi|149242532


393.46 527.98 777.03 516.91 454.92 422.37

39 30 82 60 57 32

2+ 2+ 2+ 2+ 2+ 2+

5-11 12-20 134-146 269-276 420-426 716-723

SPEPEVK ILVDRDPVK GIQITSGFFQIWR YADFLTFR YNDLLDR ALSI

gi|11497525

Photosystem I reaction center subunit XI, chloroplast precursor (PSI-L) (PSI subunit V) [Spinacia oleracea]

613.85 756.55 567.45 722.47

35 60 42 63

3+ 2+ 3+ 2+

101-118 157-171 172-186 174-186

GVEVGLAHGFLLVGPFVK EGEPSIAPALTLTGR KKQPDQLQSADGWAK QPDQLQSADGWAK

gi|3914473

Photosystem I reaction center subunit IV, chloroplast precursor (PSI-E) [Spinacia oleracea]

603.84 524.09 635.47

60 39 63

3+ 2+ 2+

35-54 55-65 82-94

AAEEAAAAPAAASPEGEAPK AAAKPPPIGPK GVGSVVAVDQDPK

gi|131178

Photosystem I subunit VII [Spinacia oleracea]

823.43 946.48

94 50

2+ 3+

7-19 20-35

IYDTCIGCTQCVR ACPTDVLEMIPWDGCK

gi|11497582

Chlorophyll a/b binding protein presusor [Oryza sativa (japonica cultivar-group)]

659.95 687.17 694.97

43 36 42

2+ 3+ 2+

83-93 150-166 155-166

WFVQAELVNGR WQDIKNPGCVNQDPIFK NPGCVNQDPIFK

gi|72580631

Photosystem I reaction center subunit III, chloroplast precursor (Light-harvesting complex I 17 kDa protein) (PSI-F) [Spinacia οleracea] Photosystem I P700 chlorophyll a apoprotein A1 [Spinacia oleracea]

674.56 843.63 688.58

527.9 516.86 454.83 422.38

30 60 57 32

2+ 2+ 2+ 2+

12-20 269-276 420-426 716-723

ILVDRDPVK YADFLTFR YNDLLDR ALSIVQGR

gi|11497525

ATP synthase CF1 beta subunit [Spinacia oleracea]

868.08 597.05 978.07 491.39 736.53 975.57 980.57 717.55

61 75 79 74 74 88 83 72

3+ 2+ 3+ 2+ 2+ 2+ 2+ 2+

23-39 76-87 110-127 179-191 218-231 249-261 262-277 278-291

IAQIIGPVLDVAFPPGK AVAMSATDGLTR IFNVLGEPVDNLGPVDTR TVLIMELINNIAK Oxidation (M) ESGVINEQNIAESK VGLTALTMAEYFR DVNEQDVLLFIDNIFR FVQAGSEVSALLGR

gi|11497535

ATP synthase CF1 alpha subunit [Spinacia oleracea]

800.03 709.07 1122.13 626.56

84 75 42 38

2+ 2+ 2+ 2+

26-41 95-107 142-162 481-491

VVNTGTVLQVGDGIAR IAQIPVSEAYLGR SVYEPLQTGLIAIDAMIPVGR TFTEEAEALLK

gi|126022791

316

Band at 580 kDa: PSI (RCI + LHCI), ATPase, PSII Core Dimer 59 2+ 108-120 LYADDSAPALAIK 77 2+ 195-209 EIIIDVPLASSLLFR 35 2+ 220 - 231 ELLNGELVDNNF


gi|19855891

gi|131187

research articles

Hydroponic Spinach Plants under Cadmium Treatment Table 1. Continued peptides identified by MS/MS subunit name

m/z

Mascot score%

charge state

start-end

Photosystem II 47 kDa protein [Spinacia oleracea]

403.03 663.90 656.71 905.95 743.34 352.62

49 51 64 83 33 30

2+ 2+ 3+ 2+ 2+ 2+

8-18 58-68 287-304 288-304 309-321 379-384


527.9 516.86 454.83 422.38

30 60 57 32

Band at 425 kDa: PSI (RCI) 2+ 12-20 ILVDRDPVK 2+ 269-276 YADFLTFR 2+ 420-426 YNDLLDR 2+ 716-723 ALSIVQGR

ATP synthase CF1 alpha subunit [Spinacia oleracea]

800.03 709.07 748.53 626.55

CP47 [Spinacia oleracea]

906.07 743.45 724.00 645.98

67 44 65 38

2+ 2+ 3+ 2+

291-307 312-324 330-350 427-437

VSAGLAENQSFSEAWSK LAFYDYIGNNPAK AGSMDNGDGIAVGWLGHPIFR AQLGEIFELDR

gi|1420852

photosystem II protein D2 [Spinacia oleracea]

614.39 774.58 521.53

56 64 40

2+ 2+ 2+

296-305 306-318 319-327

AYDFVSQEIR AAEDPEFETFYTK NILLNEGIR

gi|11497520

Apocytochrome f precursor

560.36 617.97 594.66 477.36 492.27

35 37 54 38 70

3+ 2+ 3+ 2+ 2+

132-145 146-157 221-236 237-244 313-320

IGNLSFQNYRPNKK NILVIGPVPGQK EKGGYEITIVDASNER QVIDIIPR VQLSEMNF. Oxidation (M)

gi|544122

Photosystem Q(B) protein (32 kDa thylakoid membrane protein) (Photosystem II protein D1)

714.35 730.06 730.18 657.87 429.19

45 48 47 65 70

Photosystem II 47 kDa protein [Spinacia oleracea]

403.03 671.89 656.71 905.95 743.34 352.62 723.91 645.43

49 64 33 51 65 33 51 38

3+ 2+ 3+ 2+ 2+ 2+ 2+ 2+

8-18 58-68 287-304 288-304 309-321 379-384 423-434 424-434

VHTVVLNDPGR QGMFVIPFMTR.L Oxidation (M) RVSAGLAENQSFSEAWSK VSAGLAENQSFSEAWSK LAFYDYIGNNPAK ADVPFR RAQLGEIFELDR AQLGEIFELDR

gi|11497552

Photosystem II protein D2 [Spinacia oleracea]

774.58 521.53

66 40

2+ 2+

306-318 319-327

AAEDPEFETFYTK NILLNEGIR

gi|11497520

Chlorophyll a-b binding protein, chloroplast precursor (LHCII type I CAB) (LHCP) [Spinacia oleracea]

740.44 663.51 492.37

55 39 49

Lhcb2 protein [Arabidopsis thaliana]

711.17 711.52 442.69 953.04 960.52 492.43

27 31 30 60 31 29

Major chlorophyll a/b binding protein LHCb1.2 [Spinacia oleracea]

711.51 663.51 492.37

62 39 49

Major chlorophyll a/b binding protein LHCb1.1 [Spinacia oleracea]

711.51 663.51 492.37

59 39 49

Band at 315 kDa: ATPase 75 2+ 33 2+ 110 2+ 38 3+

sequence

VHTVVLNDPGR QGMFVIPFMTR Oxidation (M) RVSAGLAENQSFSEAWSK VSAGLAENQSFSEAWSK LAFYDYIGNNPAK ADVPFR

Subunit, PSII Core, Cyt b6/f Dimer 26-41 VVNTGTVLQVGDGIAR 95-107 IAQIPVSEAYLGR 142-162 SVYEPLQTGLIAIDAMIPVGR 481-491 TFTEEAEALLK

Band at 209 kDa: PSII 2+ 17-27 3+ 239-257 2+ 258-269 2+ 313-323 3+ 324-334

Less CP43 FCDWITSTENR FGQEEETYNIVAAHGYFGR LIFQYASFNNSR VINTWADIINR ANLGMEVMHER.N Oxidation (M)

Band at 155 kDa: Trimeric LHCII 2+ 44-56 TVQSSSPWYGPDR 2+ 96-105 NRELEVIHCR 2+ 127-134 FGEAVWFK

3+ 2+ 2+ 2+ 2+ 2+

37-56 44-56 96-105 106-122 127-134 213-217

KTAGKPKNVSSGSPWYGPDR NVSSGSPWYGPDR NRELEVIHCR WAMLGALGCVFPELLAR Oxidation (M) FGEAVWFK VKEIK

Band at 90 kDa: Monomeric LHCI 2+ 44-56 NVSSGSPWYGPDR 2+ 96-105 NRELEVIHCR 2+ 127-134 FGEAVWFK 2+ 2+ 2+

44-56 96-105 127-134

NVSSGSPWYGPDR NRELEVIHCR FGEAVWFK


gi|11497552

gi|11497525

gi|126022791

gi|1709829

gi|115780

gi|4741944

gi|133917261

gi|133917263

a Each band contained one or more multiprotein complex recognized on the basis of the identified proteins. Molecular weight of the bands referred to Figure 3A.

+ LHCI), Rubisco and the dimeric PSII core. The band at 425 kDa became denser after 15 days of Cd treatment. Trypsin

digestion of this multiprotein complex gave a reduced number of peptides (see Table 1) with respect to the control, suggesting Journal of Proteome Research • Vol. 8, No. 1, 2009 317

research articles that the polypeptides from Cd treated-leaves contained modified amino acids which are not easily attacked by trypsin.46 Moreover, on the basis of the identified polypeptides (see Table 1), we revealed that many small PSI core proteins were missing and consequently hypothesized that band may correspond to an “incomplete” PSI core (RCI) (see below), deriving from the simultaneously disaggregation of PSI complexes and supercomplexes at 580 and 709 kDa, respectively. No significant variation of the band at 315 kDa, which contains the monomeric PSII core complex, one ATP-synthase subunit, and the dimeric Cyt b6/f-complex, was observed. The volume of the band at 209 kDa, containing the monomeric PSII core-less CP43, increased after 15 days of Cd treatment, probably reflecting the disaggregation of the PSII dimeric form at 580 kDa, which also started occurring at 15 days. Finally, the two bands at about 155 and 90 kDa, representing the trimeric and monomeric forms of the light-harvesting complex of PSII (LHCII), respectively, did not change significantly. Only a modest decrease of trimers was observed after 15 days of Cd treatment. Thus, all these observations suggest that PSI is the most affected complex just at the onset of Cd treatment, whereas PSII shows significant damage only after 15 days of Cd treatment. ATP-synthase and Cyt b6/f-complex are not affected by Cd. Interestingly, the semiquantitative band analysis of Cd-treated thylakoid apical leaves performed with BN profile did not show significant differences (supplementary Figure 2 in Supporting Information), indicating that the photosynthetic apparatus of these leaves was not affected. These results agree with the apical leaves [Chl]tot measurements (Figure 1A). 2D BN-SDS-PAGE. Basal leaves blue native gel lanes from control (Figure 4A lower panel) and Cd stress (Figure 4A upper panel) at 0, 5, 10 and 15 days of treatment were directly transferred and analyzed into the second denaturing 6 M urea SDS-PAGE. Here, protein components of the thylakoid membrane complexes are distributed by their molecular masses, and proteins are recognized by separated vertical lines in the 2D gel. Interestingly in the Cd-treated gels, after 15 days of Cd treatment, multimeric PSI spots, contained in the first vertical lane, disappeared completely, while the few spots present in the second vertical lane corresponded only to the to ATPase complex proteins (see rectangle of Figure 4A upper panel, day 15 map), corroborating that PSI(RCI + LHCI) and PSII dimeric core are completely degraded. This result supports the conclusion that PSI supercomplexes, PSI-RCI, and PSII dimers were completely destroyed by Cd after 15 day of treatment. Only one spot was also found in the lane corresponding to the 425 kDa band containing the PSI(RCI) complex, which was identified as PsaA/PsaB (see circle of Figure 4A upper panel, day 15 map). However, no PSI antennae and other smaller PSI core proteins were revealed by the assay. No significative variation have been observed among the 2DE maps of control sample (Figure 4A lower panel). To compare the protein patterns along the treatment, a semiquantitative analysis of the spots differentially expressed in 2D BN-SDS-PAGE was obtained by using Progenesis SameSpots software vers. 2.0 (Nonlinear Dynamics). The originality of our approach was to use a 2D BN-SDS-PAGE system for a semiquantitative analysis. A match set was created matching the proteins patterns of five replicate gels for each thylakoid extract. Five replicates were performed to achieve statistical power of 0.8 with a 2-fold change. The master map obtained comparing maps at 0-10 days of Cd treatment is shown in Figure 4B. We decided to analyze maps at 10 days because, on 318


Fagioni et al. the basis of 1D BN-PAGE analysis, 15 days of Cd treatment damaged significantly the photosytem apparatus. Eighteen proteins were found to either increase or decrease significantly (p < 0.05) during this treatment: seven proteins were upregulated (spots 1, 6, 7,10, 11, 21, 23) and 11 proteins were down-regulated (spots 8, 13, 15, 16, 17, 19, 20, 22, 25, 26, 28). Interestingly, the total spot numbers between the replicates were highly reproducible, with the standard deviation of the total mean number spots between (1.3-1.7 (see Figure 4B). Furthermore, the reproducibility of spot normalized volumes, between the replicates, was also very high. Both observations reported above indicated the 2D BN-SDS-PAGE system as a potent tool for the semiquantitative analysis of hydrophobic proteomes. Differentially expressed gel spots were subjected to an ingel tryptic digestion and the proteins contained in each were identified by mass spectrometry (Table 2). Spots 1, 6, 11 and 23, belonging to dimeric PSII core components (the band at 580 kDa in Figure 3A), were identified, respectively, as CP43, CP47, D1 and D2 proteins. The up-regulated spot 21 was identified as containing PsaA/PsaB proteins from the PSI subcomplex. Taken together, these results indicate that PSII core proteins dimer increased during the first 10 days of Cd treatment, when PSI complex and supercomplexes were observed to decrease. Other overexpressed proteins were Ferrodoxin Reductase (a free protein, attributed to spot 7), and oxygen-evolving enhancer protein 2 (OEE2) (another free protein, assigned to spot 10). On the contrary, Rubisco (assigned to spot 22) decreased during the first 10 days of Cd treatment. LHCII proteins from the monomeric PSII core complex (spots 17 and 15), the monomeric PSII core complex less CP43 (spot 19) and the trimeric LHCII (spot 20), LHCI from both PSI supercomplexes (spot 28) and PSI(RCI + LHCI) (spot 25, 26) and PsaA/PsaB (spot 8) from PSI supercomplexes, all decreased after 10 days of Cd treatment. Chromatographic Separation of Thylakoid Membranes. Since antenna proteins appeared to be mostly affected by Cd treatment, we used liquid chromatography, a method that allows better qualitative and quantitative antenna determination than 2D electrophoresis.34,35,47 Thus, thylakoid membranes extracted from basal leaves after 0, 5, 10 or 15 days of Cd treatment were loaded onto a sucrose gradient and separated into subcomplexes. Five green fractions were obtained from leaves after 5-10 days of Cd treatment, while three bands were observed from the Cd-treated sample after 15 days of treatment (Figure 5A). After 5 days of Cd treatment, the first band (B1), containing monomeric PSII antennas,42 showed a density similar to the control. The second band (B2), corresponding to Lhcb trimeric aggregates (LHCII), was fainter in Cd-treated leaves than in controls, confirming the indications by 1D BNPAGE that antenna trimerization was reduced by Cd. The third band (B3), containing the PSII core, was apparently not affected by the 5 days Cd treatment, while the two bands (B4 and B5), both containing PSI-Lhca, were strongly reduced in Cd-treated leaves compared to controls. After 15 days of Cd treatment, these two bands disappeared completely, and a new band (indicated by an asterisk in Figure 5A), with a lower molecular mass, appeared. The more intense color of this new band suggests the formation of a new subcomplex in Cd-treated leaves, in agreement with the detection of a larger band at 425 kDa observed by 1D BN-PAGE (Figure 3A), which corresponded to an “incomplete” PSI core. When this band was digested by


research articles

Figure 4. 2D BN-SDS-PAGE maps of thylakoid membrane proteins extracted from 0, 5, 10 and 15 days of both control and Cd treatment in spinach basal leaves (panels A). Panel (B) show the master map created by comparison of the 0 and 10 days maps (five replicates for each sample). This analysis was performed by Progenesis Same Spot software ver. 2.0 (Nonlinear Dynamics). The numbers shown refer to proteins identified by mass spectrometry and detailed in Table 2. The mean number of total spots number ( standard deviation is also annotated. Gels were stained with colloidal Coomassie blue. Total protein sample load: 100 µg. The lower side of panel (B), shows histograms referred to the 18 spots that were found to be differentially regulated upon Cd treatment.

trypsin, a low amount of peptides was also detected, corroborating the conclusion that modified amino acids are present in the polypeptides chain of PsaA/PsaB proteins in Cdtreated leaves.

Each ultra centrifuged band was collected with a syringe and analyzed by HPLC. Protein identification was performed by comparison of the measured molecular masses with those deduced from the DNA sequences in Cd-treated and control Journal of Proteome Research • Vol. 8, No. 1, 2009 319

research articles

Fagioni et al. a

Table 2. Proteins Identified in 2D BN-SDS-PAGE Map of Spinach Thylakoid

peptides identified by MS/MS ID

m/z

Mascot score %

major chlorophyll a/b binding protein LHCb1.2 [Spinacia oleracea]

16

711.30 663.39 528.35

62 39 49

2+ 2+ 2+

44-56 96-105 127-134

NVSSGSPWYGPDR NRELEVIHCR ELEVIHCR

gi|133917261

Chlorophyll a-b binding protein, chloroplast precursor (LHCII type I CAB) (LHCP) [Spinacia oleracea] Lhcb2 protein [Arabidopsis thaliana]

16

740.36 492.34

55 49

2+ 2+

44-56 125-132

TVQSSSPWYGPDR. FGEAVWFK

gi|115780

19

544.74 418.31 491.82 492.24

29 72 31 52

3+ 3+ 2+ 2+

43-56 94-103 96-103 125-132

STPQSIWYGPDRPK NRELEVIHSR ELEVIHSR FGEAVWFK

gi|4741944


13

740.36 492.35

55 49

2+ 2+

44-56 127-134

TVQSSSPWYGPDR. FGEAVWFK

gi|115780

Major chlorophyll a/b binding protein LHCb1.2 [Spinacia oleracea

15

711.47 824.98 663.41 960.67 983.66 492.36

77 57 38 77 50 48

2+ 2+ 2+ 2+ 2+ 2+

44-56 44-58 96-105 98-105 106-122 127-134

NVSSGSPWYGPDR NVSSGSPWYGPDRVK NRELEVIHCR ELEVIHCRGlufpyro-Glu (N-term E) WAMLGALGCVFPELLAROxidation (M) FGEAVWFK

gi|133917261


17

711.30 663.39 528.35 492.24

62 39 49 30

2+ 2+ 2+ 2+

44-56 96-105 127-134 98-105

NVSSGSPWYGPDR NRELEVIHCR ELEVIHCR FGEAVWFK

gi|133917263

light-harvesting chlorophyll a/b-binding protein [Prunus persica]

17

711.49 825.00 627.06 492.37

31 32 29 60

2+ 2+ 2+ 2+

44-56 44-58 96-105 127-134

NVSSGSPWYGPDR NVSSGSPWYGPDRVK NRELEVIHSR FGEAVWFK

gi|556367


17

740.44 663.51 492.37

55 39 49

2+ 2+ 2+

44-56 96-105 127-134

TVQSSSPWYGPDR NRELEVIHCR FGEAVWFK

gi|115780

photosystem II 44 kDa protein (CP43) [Arabidopsis thaliana]

1

713.92 740.88 491.81 902.53

70 80 40 78

2+ 2+ 2+ 3+

324-339 344-357 363-370 458-473

LGANVGSAQGPTGLGK SPTGEVIFGGETMR APWLEPLR GIDRDFEPVLSMTPLN

gi|7525029

CP47 [Spinacia oleracea]

6

656.34 723.95 729.36 751.80 352.69 724.45 645.93

63 45 38 29 33 51 65

3+ 3+ 3+ 2+ 2+ 2+ 2+

290-307 330-350 361-381 362-381 362-381 382-387 426-437

RVSAGLAENQSFSEAWSK AGSMDNGDGIAVGWLGHPIFR RMPTFFETFPVVLIDGDGIVR MPTFFETFPVVLIDGDGIVR ADVPFR RAQLGEIFELDR AQLGEIFELDR

gi|1420852

Chain A, Refined Crystal Structure Of Spinach Ferredoxin Reductase At 1.7 Angstroms Resolution: Oxidized, Reduced, And 2′Phospho-5′-Amp Bound States

7

737.53 915.5

39 49

2+ 2+

1-15 1-18

gi|157831108

807.98 669.52 947.52

79 60 36

2+ 2+ 2+

94-109 118-129 130-146

QIASDVEAPPPAPAK QIASDVEAPPPAPAKVEKGlnf pyro-Glu (N-term Q) LYSIASSALGDFGDAK LIYTNDAGETIK GVCSNFLCDLKPGAEVK

690.48

26

2+

265-275

DNTYVYMCGLK

Photosystem I P700 Apoprotein A1 [Nicotiana silvestri]

8

393.46 528.00 493.52 517.01 455.15

39 30 29 60 57

2+ 2+ 3+ 2+ 2+

5-11 12-20 233-244 269-276 420-426

SPEPEVK ILVDRDPVK EIPLPHEFILNR YADFLTFR YNDLLDR

gi|225198

10

795.48 899.05 680.46

49 61 73

3+ 2+ 2+

130-150 216-233 255-267

YEDNFDATSNLSVLVQPTDKK TADGDEGGKHQVIAATVK KFVESATSSFSVA

gi|131392

subunit name

Oxygen-evolving enhancer protein 2, chloroplast precursor (OEE2) (23 kDa subunit of oxygen evolving system of photosystem II) (OEC 23 kDa subunit) (23 kDa thylakoid membrane protein) [Spinacia oleracea]

320


charge state

start-end

sequence


research articles

Hydroponic Spinach Plants under Cadmium Treatment Table 2. Continued peptides identified by MS/MS ID

m/z

Mascot score %


20

711.30 663.39 528.35

59 39 49

2+ 2+ 2+

44-56 96-105 127-134

NVSSGSPWYGPDR NRELEVIHCR ELEVIHCR

gi|133917261

Chlorophyll a-b binding protein, chloroplast prescursor (LHCII type I CAB) (LHCP) [Spinacia oleracea]

26

740.44 663.51 492.37

30 39 49

2+ 2+ 2+

44-56 96-105 127-134

TVQSSSPWYGPDR NRELEVIHCR FGEAVWFK

gi|115780

Photosystem Q(B) protein (32 kDa thylakoid membrane protein) (Photosystem II protein D1)

11

714.35 730.06 730.18 657.87 429.19

55 50 85 39 51

2+ 3+ 2+ 2+ 3+

17-27 239-257 258-269 313-323 324-334

FCDWITSTENR FGQEEETYNIVAAHGYFGR LIFQYASFNNSR VINTWADIINR ANLGMEVMHER.N Oxidation (M)

gi|1709829

RCA (RUBISCO ACTIVASE) [Arabidopsis thaliana]

22

710.95 511.36 489.59 456.27 369.25 631.33 481.80 605.05 456.79 478.69 607.42 614.94 773.98 927.54

50 45 66 43 70 38 84 37 59 41 39 66 71

2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+

22-32 33-41 147-159 188-194 195-201 218-227 228-236 237-252 296-303 320-334 335-350 436-446 451-463 464-479

LNYYTPEYETK DTDILAAFR TFQGPPHGIQVER AVYECLR GGLDFTK FLFCAEAIYK SQAETGEIK GHYLNATAGTCEEMMK Oxidation (M) AMHAVIDR LSGGDHVHAGTVVGK LEGDRESTLGFVDLLR DLAVEGNEIIR WSPELAAACEVWK EITFNFPTIDKLDGQD

gi|18405145

Photosystem I P700 chlorophyll a apoprotein A2 (Psa-B) (PSI-B)

21

493.09 425.79 838.97 693.97 716.54 737.93 449.54

35 80 54 35 61 30 36

2+ 2+ 2+ 2+ 2+ 2+ 2+

8-20 303-314 397-410 452-469 470-490 552-564 685-692

FSQGLAQDPTTR FSQGLAQDPTTRR DLLEAHIPPGGR DYNPEQNEDNVLAR QILIEPIFAQWIQSAHGK TSYGFDVLLSSTSGPAFNAGR DFGYSFPCDGPGR

gi|131149

Photosystem I light-harvesting chlorophyll a/b binding protein

25

711.49 825.00 627.06 492.37

72 45 66 35

2+ 2+ 2+ 2+

44-56 44-58 96-105 127-134

NVSSGSPWYGPDR NVSSGSPWYGPDRVK NRELEVIHSR FGEAVWFK

gi|483723

PSI type III chlorophyll a/b-binding protein [Arabidopsis thaliana]

28

646.41 815.62 823.55 490.39

41 51 35 32

2+ 2+ 2+ 2+

95-105 106-122 171-182 183-190

WLAYGEIINGR FAMLGAAGAIAPEILGK LQDWYNPGSMGK Oxidation (M) QYFLGLEK Glnfpyro-Glu (N-term Q)

gi|430947

Photosystem II D2 protein (Photosystem Q(A) protein) (PSII D2 protein)

23

613.80 774.46

56 66

2+ 2+

296-305 306-318

AYDFVSQEIRAAEDPEFETFYTK AAEDPEFETFYTK

gi|131296

subunit name

a

charge state

start-end

sequence


Spot ID number related to numbers shown in Figure 4B.

leaves, as shown elsewhere.47,48 B1 and B2 bands were mixed together because in this way they represented the total PSII antenna. After 5 days of Cd treatment, Lhcb1.1 isomers were completely suppressed while the other isomeric forms of Lhcb1, and Lhcb2 and Lhcb3 were slightly affected (Figure 5B). Interestingly, the chromatogram of B4 and B5 (again mixed together to represent PSI components) showed that all PSI antennae disappeared after 5 days of Cd treatment, while the PsaA and PsaB fractions increased over the same time course (Figure 5C). This result confirmed the electrophoretic data showing that an “incomplete” PSI core accumulated in Cdtreated leaves, as a consequence of PSI antenna supercomplexes destruction. Chromatographic separation was also performed in leaves recovering from Cd stress. Interestingly, upon removal of Cd, a resynthesis of the Lhcb1.1 antenna was observed just after three days of the reconstitution experiment, but its amount

increases significantly upon Zn2+ addition to the irrigation water (Figure 5D). This result suggests that, contrary to Chl resynthesis (see Figure 1), Lhcb1.1 gene expression is strongly Zn-dependent. Expression Levels of Lhcb1 Genes. The accumulation of Lhcb1.1 transcripts during prolonged, continuous Cd treatment of spinach plants was investigated by Real Time PCR, using RNA purified from basal leaves exposed to different periods of Cd treatment. To discriminate the Lhcb1.1 isoforms, specific primer pairs were designed,49 exploiting some differences occurring in for the coding sequence. Primer pairs specificity was tested by PCR amplifications on the corresponding Lhcb1 cDNA clone. The results of RT Real Time PCR experiments are shown in Figure 6. Interestingly, Cd treatment caused a clear evident decrease in transcript accumulation of the Lhcb1.1 mRNA over time. This effect was particularly dramatic during the first 10 days of Cd treatment, as shown by the steeper slope Journal of Proteome Research • Vol. 8, No. 1, 2009 321

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Fagioni et al. faster decay rate was observed for basal Cd-treated leaves, whereas control and apical leaves showed a similar behavior. Thus, Cd treatment seems to affect the reduction rate of TyrD+ only in basal leaves.

Discussion

Figure 5. Separation onto a sucrose gradient of thylakoid MPCs in 5 different fractions (B1-B5) in control (0 day) and stressed (5, 10, and 15 days of Cd) spinach basal leaves (A). Asterisk indicated a new band appeared in 15 day tube. In panels (B and C) the HPLC analysis of each thylakoid subcomplex in leaves after 0 (black lines) or 5 (gray lines) days of Cd treatment is shown. Panel D reports the Lhcb1.1 resynthesis upon Cd2+ removal or upon addition of Zn2+.

of transcript level decay in Figure 6. The negative effect of Cd was partially and quickly recovered just after 3 days of Cd removal from plants growth for 30 days in the presence of Cd. The recovery was significantly higher than when high Zn2+ ions concentration were added to the irrigation water instead of Cd, indicating a role of this metal ion on Lhcb1.1 gene expression. ESR spectroscopy. To study how Cd2+ affects electron transfer reactions, intact thylakoid membranes (control and both Cd treated basal and apical leaves at 30 days) were subjected to continuous strong white light illumination at room temperature and ESR spectra were recorded directly. The TyrD+ signal was first monitored, but no significant differences in the shape of the spectrum were revealed between the three samples (data not shown). By contrast, interesting information was obtained by following the kinetics of TyrD+ formation. In this experiment, the size of the TyrD+ peak was monitored continuously allowing precise determination of the reaction kinetics (Figure 7). It can be noticed that the TyrD+ signal reached a plateau within seconds after the onset of irradiation; this type of kinetics is very common for free radicals in solution and is normally due to reactions involving two radicals. Surprisingly, when the light was turned off, the TyrD+ signal started to disappeared but with different rates. In particular, a 322


Pigment analysis reveals strong inhibition of Chl only in basal leaves. It has been suggested that Cd induced leaf chlorosis might indicate impairment of the Mg2+ insertion into protoporphyrinogen,50 or direct Chl destruction as consequence of Mg ion substitution in both Chl a and b.51 Interestingly, in our experiment, basal leaves of plants treated with Cd showed a clear and very rapid inhibition of Chl a, followed by a less evident inhibition of Chl b. Perhaps also Cd induced impairment of iron uptake by roots4 may play a role in determining leaf chlorosis. However, our previous investigation in irondepleted spinach plants42 showed that Chl b prevalently decreased during the first 10 days, contrary to what we now observe upon Cd treatment, suggesting that impairment of iron uptake by roots4 plays a minor role and different mechanisms are involved. It has been also proposed that Chl reduction might be caused by a direct interference of Cd with enzymes of the Chl biosynthesis pathways4 or by Cd interference with the correct assembly of the pigment-protein complexes of the photosystems.4,19 If this is the case, the effect should be reversible. Upon Cd removal, we observed a quick resynthesis of both Chl, indicating that inhibition of Chl biosynthesis is reversible. Zinc may further alleviate Cd induced toxicity,52 but in the specific case of Chl damage, we found that recovery is not dependent on Zn presence. This agrees with the hypothesis that the effect of Cd2+ on the biosynthesis of Chl may occur by affecting the protochlorophyllide reductase53 which contains oxygen-tolerant Fe-S clusters.54 It is well-known that Cd triggers the inhibition of root Fe(III) reductase,9 which leads to a decreased shoot iron content;55 therefore, a formation of zeaxanthin was expected even at 300 µE m-2 s-1, similarly to what was observed in spinach plants grown in iron deficiency.42 Contrary to our expectation, no formation of zeaxanthin and antheraxanthin was observed upon Cd treatment, in agreement to what was reported by other authors10,56 that a modification of ferredoxin activity by Cd causes the inhibition of zeaxanthin formation. The molecular mechanism was attributed to the interaction of Cd with a cysteine residue located in motif I of Ferredoxin NADP+ oxidoreductase (FNR). Spinach ferredoxin treated with Cd showed not iron but Cd into iron-sulfur clusters, indicating a replacement between two ions.12 Our proteomic investigation showed an overexpression of Ferredoxin in basal leaves, as a possible reaction of plant to its reduced activity. Finally, a remarkably increase of lutein was observed, especially after 15 days of Cd treatment. Lutein stimulation may indicate the attempt of plant to activate a defensive mechanism against ROS that likely accumulates because of photosynthesis inhibition over time.17,57 We did not measure ROS accumulation in Cd-treated basal leaves, but it is well-known that ROS are produced during Cd treatment.14,15 ROS may cause amino acid oxidation, in particular, the oxidation of arginine and lysine into glutamic-semialdehyde and 2-amino-adipic-semialdehyde, respectively,58 which can give cross-links reacting with other Lys, Cys or His residues.59 Thus, it is not surprising that trypsin digestion of proteins, contained into electrophoretic gel bands or in ultracentrifuged bands, is influenced, as observed in our experiment. To this


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Figure 6. Quantitative mRNA transcript level of one isoform (Lhcb1.1) of spinach light harvesting complex gene in leaves of plants that were treated with Cd for 5, 10, 15, 30 and 33 days. * (P < 0.01) statistically different from day 0 value. mRNA transcripts of leaves of plants upon Cd removal or upon addition of Zn are also shown. # (P < 0.05, referred to Cd free value) statistically different from 33 day value, + (P < 0.001, referred to Zn value) statistically different from 33 day value. One-way ANOVA test.

Figure 8. Representation of semiquantitative MPCs changes observed in spinach basal leaves upon Cd treatment. The diagram was deduced by the combination of the data from 1D BN-PAGE and 2D BN-SDS-PAGE. Grayscale indicates the variation of complex under stress from 0% (white) to 100% (black).

Figure 7. Kinetics of the formation of TyrD+ upon illumination of thylakoids from Cd-treated leaves at 30 days. The kinetics of TyrD+ was measured by monitoring the ESR signal at 3465 G and the intensity of light was 3300 µmol m-2 s-1. The light as turned off at the point indicated by the arrow (V).

regard, it may be hypothesized that lutein overproduction in Cd stressed leaves may represent a defensive mechanism of plants toward ROS production. Proteomic Analysis Reveals High Sensitivity of PSI to Cd and only Minor Effects on PSII. Figure 8, obtained by the combination of the 1D BN-PAGE and 2D BN-SDS-PAGE data, provides a schematic representation of the photosynthetic complexes quantitative changes observed in spinach basal leaves upon Cd treatment. Interestingly, the amount of Cytochrome b6/f and ATP-synthase does not change during the 15 day Cd treatment, suggesting that only MPCs containing Chl are sensitive to Cd. It can be also seen that Cd drastically reduced the amount of PSI protein just at the onset of Cd exposure: both the functional monomeric PSI(RCI + LHCI) and the multimeric aggregates (PSI supercomplexes) decrease, disappearing after 15 days. An “incomplete” PSI(RCI) multi-

protein complex increased over time, indicating a PSI supercomplex degenerative process. Interesting, PSI is unaffected by Cd in Synechocystis PCC 6803,60 which may indicate a different sensitivity to Cd in the higher plants and algae. In our experiments, chromatografic and electrophoretic analysis revealed that Lhca decreased significantly already at the onset of Cd stress, and disappeared within 10-15 days of Cd treatment, simultaneously with the accumulation of an “incomplete” PSI(RCI) subcomplex containing PsaA and PsaB with modifiedaminoacids,andlikelyinducedbyROSaccumulation.14,61 It might be speculated that early reduction of PSI antennae reflects the early disappearance of Chl a in Cd-treated leaves, since PSI antennae mainly contain this Chl type.62 Subsequently, further reduction of PSI complexes, as observed after 10-15 days of Cd treatment, might be caused by formation of oxygen radicals. In Synechocystis PCC 6803, Cd altered the oxidation kinetics of P700, and affected the available electron acceptor pool of P700.63 Thus, early events associated to Chl a degradation and perturbing PSI protein assembly may reduce the electron flow through PSI. Consequently, more reactive oxygen species can be produced by alternative electron flow, such as direct photoreduction of oxygen, which destroys Lhca antennae and attacks core complexes (RCI) of this photosystem. Apparently, the PSII complexes are less affected than the PSI complexes, at least during the first 10-15 days. Indeed, as shown in Figure 8, the amount of the PSII complexes change in response to the PSI decrement. In particular, the amount of PSII core dimer increased during the first 10 days, falling down Journal of Proteome Research • Vol. 8, No. 1, 2009 323

research articles during the subsequent 5 days. The PSII “core less” CP43 complex also increased, probably as consequence of the PSII core dimer reduction. It is our opinion that the PSII core dimer fluctuations is a physiological response of plants to the PSI complexes deactivation. However, monomeric PSII core level remains constant, contrary to the trimeric LHCII which decreases. Chromatography of PSII antenna showed a specific reduction of Lhcb1.1 isomer just after the onset of treatment, while the other PSII antenna components remained unchanged. The rapid Lhcb1.1 mRNA resynthesis, as well as the rapid resynthesis of Lhcb1.1 antenna, upon addition of Zn ions indicated that a specific Zn-finger trascription factor may be involved in Lhcb1.1 gene expression, and that Cd replacement of Zn may specifically affect this protein. However, upon Cd treatment in plants, besides complex rearrangements, single proteins also resulted overexpressed, such as Ferredoxin, discussed above, and oxygen evolving complex (OEC) 23 kDa subunit. Regarding the last protein, it is well-known that Cd2+ can exchange, with high affinity in a slow reaction, for the Ca2+ cofactor in Ca/Mn cluster that constitutes the oxygen-evolving center.64 Interestingly, the 23 kDa subunit is known to allow PSII to evolve oxygen under limiting Ca2+,65 suggesting that this polypeptide acts as concentrator of these ions. The substitution of the essential Ca2+ with a nonfunctional Cd2+ cation could promote its overexpression, as reported here. In line with a perturbation of OEC activity, ESR analysis showed a faster decay rate of TyrD+ radical in Cd-treated basal leaves compared to control and apical leaves. In fact, the TyrD+ is thought to oxidize the Mn through electron transfer equilibria between the intervening cofactors, that is, TyrZ and the Chl P. Consequently, a more rapid loss of YD+ signal may influence the electron transfer between YD+ and Mn complex (S0YD+ f S1YD).66 In the case of Ferredoxin NADP-reductase, its oversynthesis may be needed to sustain the production of ATP, NADH and NADPH to be used in the synthesis of 2-oxoglutarate and phosphoglycerate, in turn producing amino acids and Cd chelating molecules, as the phytochelatins.67 We collected evidence from proteomic assays of a strong, negative effect of Cd on Rubisco protein level. In Clamydomonas, the most drastic effect of Cd treatment is the reduced abundance of both large and small subunits of Rubisco, in correlation with several other enzymes involved in photosynthesis.50 In Cd-treated cyanobacterial spheroplasts, Rubisco activity also decreased to 40% of control level, and in Cd-treated spinach chloroplasts Rubisco dropped to only 2.5% of control level,63 indicating that Cd induced severe damage to Rubisco also occurs in higher plants. In fact it was suggested that Cd could irreversibly bind to SH-groups of this enzyme, lowering its activity in vivo.68 Recently, in Cyanobacteria, it was reported that Cd might inactivate different metal-containing enzymes, including carbonic anhydrase, by replacing Zn, which would explain a rapid and almost full inhibition of the photosynthetic activity of these organisms.63 General Patterns of Cd Toxicity in Photosynthetic Apparatus and Defensive Mechanisms. Upon Cd treatment, complexes without Chl are not affected, indicating that Chl reduction is the main Cd damage effect. The specific Chl a reduction induces a progressive PSI antenna reduction, with the consequently disappearance of main proteic supercomplexes. No inhibition of a specific PSII protein was observed, beside a slight PSII core dimer increase and a significant accumulation of the monomeric form CP43 less between 9-15 324


Fagioni et al. days (see Figure 8). Interestingly, also PSII antennae, which were not completely inhibited by Cd treatment, were not found to be rearranged, shifting the monomer-trimer equilibrium, as previously observed in iron-deficient leaves.42 This suggests that plants do not successfully operate alternative strategies to cope with Cd effect at protein levels. Cd-treated leaves were also surprisingly unable to synthesize zeaxanthin, as revealed in spinach plants growth in iron deficiency,42 the best described mechanism for the quenching of damaging reactive oxygen species in leaves.55 Lutein was overproduced in Cd stressed leaves, probably because it has the specific property of quenching harmful Chl triplet by binding at site L1 of the major LCHII complex, thus, preventing ROS formation.69 However, the general reduction of proteins in Cd-treated leaves appears to be reversible once Cd treatment is stopped. This observation might support the hypothesis that Cd affects the photosynthetic apparatus only by randomly substituting other ions (e.g., Zn, Fe or Ca) into prosthetic groups of some proteins, as observed by other studies.10,14 In line, the clear and fast resynthesis of Lhcb1.1 mRNA, and consequentely the Lhcb1.1 antenna, once Cd is removed and Zn ions were added to hydroponical plants, further supports the hypothesis that Zn may be removed by Cd from some transcription factors. To this regard, it has been documented that Zn may replace Cd in complexes with cysteine.70,71 Binding of Zn and Cd in the same sites was also observed in bacterial photosynthetic reaction centers.72 Zinc appeared to reduce Cd toxicity on FNR activity, suggesting that Cd and Zn exchange in the ligands located near the quinone binding site.73 In conclusion, we surmise, based on the results of our experimentation, that Cd toxic action is mainly aspecific, probably replacing other metal ions inside proteins10,14,51 and over time by producing ROS.23 Cd affects significantly and reversibly the photosynthetic apparatus of basal leaves, while the apical leaves, where the photosynthetic apparatus is not damaged, provide most of the energy indispensable for plants survival. Furthermore, an overproduction of phytochelatins (PCs), which allows an intracellular chelation of Cd and its compartmentalization into vacuole,74 was recorded only in the basal leaves and not inside apical leaves (paper in preparation). This limits the circulation of free Cd to apical leaves and may represent a detoxification strategy adopted by higher plants, a general mechanism that is also implemented to exclude toxic anions such as Cl-.75 Abbreviations: ACN, acetonitrile; BN, blue native; N, native; LP, liquid phase; DDM, n-dodecyl-β-D-maltoside; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; FA, formic acid; LHC, light-harvesting complex; PAGE, polyacrylamide gel electrophoresis; PS, Photosystem; RP-HPLC, reversed-phasehigh performance liquid chromatography; SDS, sodium dodecyl sulfate; MPCs, multiprotein complexes; Chl, chlorophyll; ESR, electron-spin-resonance; PCR, polymerase chain reaction.

Acknowledgment. We gratefully acknowledge Dr. Sara Rinalducci (Tuscia University) for her invaluable assistance. This work was financially supported by grants from the Italian Ministry for University Research (MIUR-PRIN 2006) and GENZOT. Supporting Information Available: Time course of total Chl concentrations measured in the thylakoid membranes of control spinach plants, and Blue Native PAGE band volumes time course of thylakoid membrane protein complexes ex-

Hydroponic Spinach Plants under Cadmium Treatment tracted from apical leaves of Cd-treated plants. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Sanita` di Toppi, L.; Gabbrielli, R. Response to cadmium in higher plants. Environ. Exp. Bot. 1999, 41, 105–130. (2) Mishra, S.; Srivastava, S.; Tripathi, R. D.; Govindarajan, R.; Kuriakose, S. V.; Prasad, M. N. V. Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiol. Biochem. 2006, 44, 25–37. (3) Perfus-Barbeoch, L.; Leonhardt, N.; Vavasseur, C.; Forestier, C. Heavy metal toxicity: cadmium permeates through cadmium channels and disturbs the plant water status. Plant J. 2002, 32, 539–548. (4) Baryla, A.; Carrier, P.; Frank, F.; Coulomb, C.; Sahut, C.; Havaux, M. Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth. Planta 2001, 212, 696–709. (5) Masahiro, I.; Satoka, N.; Hiroshi, T.; Masanori, J.; Tetsuo, M. Different characteristics of roots in the cadmium-tolerance and Cd-binding complex formation between mono- and dicotyledonous plants. J. Plant Res. 1994, 107, 201–207. (6) Clemens, S. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 2006, 88, 1707–1719. ´ .; Fodor, F.; Cseh, E.; Varga, A.; Za´ray, G.; Zolla, L. (7) Sa´rva´ri, E Relationship between changes in ion content of leaves and chlorophyll- protein composition in cucumber under Cd and Pb stress. Z. Naturforsch. 1999, 54c, 746–753. (8) Roth, U.; Von Roepenack-Lahay, E.; Clemens, S. Proteome changes in Arabidopsis thaliana roots upon exposure to CdCd2+. J. Exp. Bot. 2006, 57, 4003–4013. (9) Alcańtara, E.; Romera, F. J.; Can ˜ ete, M.; De La Guardia, M. D. Effects of heavy metals on both induction and function of root Fe(III) reductase in Fe-deficient cucumber (Cucumis sativus L.) plants. J. Exp. Bot. 1994, 45, 1893–1898. (10) Grzyb, J.; Waloszek, A.; Latowski, D.; Wieˆckowski, S. Effect of cadmium on ferredoxin:NADP+ oxidoreductase activity. J. Inorg. Biochem. 2004, 8, 1338–1346. (11) Vallee, B. L. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990, 29, 5647–5659. (12) Iametti, S.; Uhlmann, H.; Sala, N.; Bernhardt, R.; Raggi, E.; Bonomi, F. Reversible, non-denaturing metal substitution in bovine adrenodoxin and spinach ferredoxin and the different reactivities of [2Fe-2S]-cluster-containing proteins. Eur. J. Biochem. 1996, 239, 818–826. (13) Bouckaert, J.; Wyns, L.; Loris, R. Zinc/calcium and cadmium/ cadmium-constituted Concanavalin A.: interplay of metal binding, pH and molecular packing. Acta Crystalogr., D 2000, 56, 1569– 1576. (14) Watanabe, M.; Henmi, K.; Ogawa, K.; Suzuki, T. Cadmiumdependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis. Comp. Biochem. Physiol., Part C 2003, 134, 227– 234. (15) Smeets, K.; Cuypers, A.; Lambrechts, A.; Semane, B.; Hoet, P.; Van Laere, A.; Vangronsveld, J. Induction of oxidative stress and antioxidative mechanisms in Phaseolus vulgaris after Cd application. Plant. Physiol. Biochem. 2005, 43, 437–444. (16) Somashekaraiah, B. V.; Padmaja, K.; Prasad, A. R. K. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): involvement of lipid peroxides in chlorophyll degradation. Physiol. Plant. 1992, 85, 85–89. (17) Pietrini, F.; Iannelli, M. A.; Pasqualini, S.; Massacci, A. Interaction of cadmium with glutathione and photosynthesis in developing leaves and chloroplasts of Phragmites australis (Cav.) Trin. ex Steudel. Plant. Physiol. 2003, 133, 829–837. (18) Haag-Kerwer, A.; Schfer, H. J.; Heiss, S.; Walter, C.; Rausch, T. Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effects on photosynthesis. J. Exp. Bot. 1999, 50, 1827–1835. (19) Szalontai, B.; Horvath, L. I.; Debreczeny, M.; Droppa, M.; Horvath, G. Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy. Photosynth. Res. 1999, 61, 241–252. (20) Atal, N.; Saradhi, P. P.; Mohanty, P. Inhibition of the chloroplast photochemical reactions by treatment of wheat seedlings with low concentrations of cadmium: analysis of electron transport activities and changes in fluorescence yield. Plant Cell Physiol. 1991, 32, 943–951.

research articles (21) Siedlecka, A.; Baszyn ˜ ski, T. Inhibition of electron flow around photosystem I in chloroplast of Cd-treated plants is due to Cdinduced iron deficiency. Physiol. Plant. 1993, 87, 199–202. (22) Prasad, M. N. V. Cadmium toxicity and tolerance in vascular plants. Env. Exp. Bot. 1995, 35, 525–545. (23) Kieffer, P.; Dommes, J.; Hoffmann, L.; Hausman, J. F.; Renaut, J. Quantitative changes in protein expression of cadmium-exposed poplar plants. Proteomics 2008, 8, 2514–2530. (24) Hoagland, D. R.; Arnon, D. I. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 1950, 347, 1–32. (25) Susıń, S.; Abiań, J.; Peleato, M. L.; Sańchez-Baeza, J.; Abadıá, A.; Gelpı´, E.; Abadıá, J. Flavin excretion from roots of iron deficient sugar beet (Beta vulgaris L.). Planta 1994, 193, 514–519. (26) Nishimura, M.; Graham, D.; Akazawa, T. Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts. Plant Physiol. 1976, 58, 309–314. (27) Dudley, J. W. Number of chloroplasts in the guard cells of inbred lines of tetraploid and diploid sugar beets. Agron. J. 1958, 50, 169– 170. (28) Ku ¨ gler, M. J.; Ja¨nsch, L.; Kruft, V.; Schmitz, U. K.; Braun, H. P. Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). Photosynth. Res. 1997, 53, 35–44. (29) Soursa, M.; Regel, R. E.; Paakkarinen, V.; Battchikova, N.; Herrmann, R. G.; Aro, E. M. Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ. Eur. J. Biochem. 2004, 271, 96–107. (30) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–686. (31) Candiano, G.; Bruschi, M.; Musante, L.; Cantucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25, 1327–1333. (32) Karp, N. A.; Spencer, M.; Lindsay, H.; O’Dell, K.; Lilley, K. S. Impact of replicate types on proteomic expression analysis. J. Proteome Res. 2005, 4, 1867–71. (33) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850–858. (34) Zolla, L.; Rinalducci, S.; Timperio, A. M.; Huber, C. G. Proteomics of light-harvesting proteins in different plant species: analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I. Plant Physiol. 2002, 130, 1938– 1950. (35) Zolla, L.; Timperio, A. M.; Walcher, W.; Huber, C. G. Proteomics of light-harvesting proteins in different plant species: analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem II. Plant Physiol. 2003, 131, 198– 214. (36) Croce, R.; Zucchelli, G.; Garlaschi, F. M.; Jennings, R. C. Thermal broadening study of the antenna chlorophylls in PSI-200, LHCI, and PSI core. Biochemistry 1998, 37, 17355–17360. (37) Johnson, G.; Scholes, J. D.; Horton, P.; Young, A. J. Relationships between carotenoid composition and growth habit in British plant species. Plant Cell Environ. 1993, 16, 681–686. (38) Gilmore, A. M.; Yamamoto, H. Y. Dark induction of zeaxanthindependent nonphotochemical fluorescence quenching mediated by ATP. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1899–1903. (39) Val, J.; Abadıá, J.; Heras, L.; Monge, E. Higher plant photosynthetic pigment analysis. Determination of carotenoids and chlorophylls by HPLC. J. Micronutr. Anal. 1986, 2, 305–312. (40) Lakshminarayana, R.; Raju, M.; Krishnakantha, T. P.; Baskaran, V. Determination of major carotenoids in a few indian leafy vegetables by high-performance liquid chromatography. J. Agric. Food Chem. 2005, 53, 2838–2842. (41) Za´ray, G.; Dao Thi Phuong, D.; Varga, I.; Kańtor, T.; Cseh, E.; Fodor, F. Influences of lead contamination and complexing agents on the metal uptake of cucumber. Microchem. J. 1995, 51, 207–213. (42) Timperio, A. M.; D’Amici, G. M.; Barta, C.; Loreto, F.; Zolla, L. Proteomic, pigment composition, and organization of thylakoid membrane in iron deficient-plant spinach leaves. J. Exp. Bot. 2007, 58, 3695–3710. (43) Ciambella, C.; Roepstorff, P.; Aro, E. M.; Zolla, L. A proteomic approach for investigation of photosynthetic apparatus in plants. Proteomics 2005, 5, 746–757. (44) Aro, E. M.; Soursa, M.; Rokka, A.; Allahverdiveva, Y.; Paakkarinen, V.; Saleem, A.; Battchikova, N.; Rintamaki, E. Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes. J. Exp. Bot. 2005, 56, 347–356. (45) D’Amici, G. M.; Timperio, A. M.; Zolla, L. Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel

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(59) (60) (61)

326

electrophoresis: a potent tool for native membrane multiprotein complex separation. J. Proteome Res. 2008, 7, 1326–1340. Dalle Donne, I.; Scaloni, A.; Butterfield, D. A. In Redox Proteomics from Protein Modifications to Cellular Dysfunction and Disease 1st ed.; Wiley-Interscience: Hoboken, NJ, 2006. Zolla, L.; Rinalducci, S.; Timperio, A. M.; Huber, C. G.; Righetti, P. G. Intact mass measurements for unequivocal identification of hydrophobic photosynthetic photosystem I and II antenna proteins. Electrophoresis 2004, 25, 1353–1366. Whitelegge, J. P.; Laganowsky, A.; Nishio, J.; Souda, P.; Zhang, H.; Cramer, W. A. Sequencing covalent modifications of membrane proteins. J. Exp. Bot. 2006, 57, 1515–1522. Rea, G.; Volpicella, M.; De Leo, F.; Zolla, L.; Gallerani, R.; Ceci, L. R. Characterization of three members of the multigene family coding for isoforms of the chlorophyll a/b binding protein Lhcb1 in spinach. Physiol. Plant 2007, 130, 167–176. Gillet, S.; Decottignies, P.; Chardonnet, S.; Le Marećhal, P. Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach. Photosynth. Res. 2006, 89, 201– 211. Ku ¨pper, H.; Ku ¨pper, F.; Spiller, M. In situ detection of heavy metal substituted chlorophylls in water plants. Photosynth. Res. 1998, 58, 123–133. Aravind, P.; Prasad, M. N. V. Zinc protects chloroplasts and associated photochemical functions in cadmium exposed Ceratophyllum demersum L., a freshwater macrophyte. Plant Sci. 2004, 166, 1321–1327. Stobart, A. K.; Griffiths, W. T.; Ameen-Bukhari, I.; Sherwood, R. P. The effects of CdCd2+ on the biosynthesis of chlorophyll in leaves of barley. Physiol. Plant 1985, 63, 293–298. Nomata, J.; Ogawa, T.; Kitashima, M.; Inoue, K.; Fujita, Y. NBprotein (BchN-BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe-S clusters. FEBS Lett. 2008, 582, 1346–1350. Fodor, F.; Ga´spa´r, L.; Morales, F.; Gogorcena, Y.; Lucena, J. J.; Cseh, ´ . Effects of two iron sources on E.; Kro¨pfl, K.; Abadıá, J.; Sa´rva´ri, E iron and cadmium allocation in poplar (Populus alba) plants exposed to cadmium. Tree Physiol. 2005, 25, 1173–1180. Latowski, D.; Kruk, J.; Strzałka, K. Inhibition of zeaxanthin epoxidase activity by cadmium ions in higher plants. J. Inorg. Biochem. 2005, 99, 2081–2087. Iannelli, M. A.; Pietrini, F.; Fiore, L.; Petrilli, L.; Massacci, A. Antioxidant response to cadmium in Phragmites australis plants. Plant Physiol. Biochem. 2002, 40, 977–982. Requena, J. R.; Chao, C. C.; Levine, R. L.; Stadtman, E. R. Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of protein. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 69–74. Davies, M. J. Singlet oxygen-mediated damage to proteins and its consequences. Biochem. Biophys. Res. Commun. 2003, 305, 761– 770. Tu ` mova´, E.; Sofrova´, D. Response of intact cyanobacterial cells and their photosynthetic apparatus to Cd 2+ ion treatment. Photosynthetica 2002, 40, 103–108. Lemaire, S.; Keryer, E.; Stein, M.; Schepens, I. I.; Issakidis-Bourguet, E.; Ge´rard-Hirne, C.; Miginiac-Maslow, M.; Jacquot, J. P. Heavy-


Fagioni et al.

(62) (63)

(64) (65)

(66) (67)

(68) (69)

(70)

(71)

(72)

(73) (74) (75)

metal regulation of thioredoxin gene expression in Chlamydomonas Reinhardtii. Plant Physiol. 1999, 120, 773–778. Jansson, S.; ersen, B.; Scheller, H. V. Nearest-neighbor analysis of higher-plant photosystem I holocomplex. Plant. Physiol. 1996, 112, 409–420. Sas, K. N.; Kovaćs, L.; Zsı´ros, O.; Gombos, Z.; Garab, G.; Hemmingsen, L.; Danielsen, E. Fast cadmium inhibition of photosynthesis in cyanobacteria in vivo and in vitro studies using perturbed angular correlation of Y-rays. J. Biol. Inorg. Chem. 2006, 11, 725– 734. Sigfridssom, K. G. V.; Berna´t, G.; Mamedov, F.; Styring, S. Molecular interference od CdCd2+ with Photosystem II. Biochim. Biophys. Acta 2004, 1659, 19–31. Ghanotakis, D. F.; Topper, J. N.; Babcock, G. T.; Yocum, C. F. Water soluble 17-kDa and 23-kDa polypeptides restore oxygen evolution activity by creating a high affinity binding site for CaCd2+ on the oxidizing side of photosystem II. FEBS Lett. 1984, 170, 169–173. Styring, G.; Rutherford, A. W. In the oxygen-evolving complex of Photosystem II the S0 state is oxidized to the S1 state by D+ (signal IIslow). Biochemistry 1987, 26, 2401–2405. Sarry, J. E.; Kuhn, L.; Ducruix, C.; Lafaye, A.; Junot, C.; Hugouvieux, V.; Jourdain, A.; Bastien, O.; Fievet, J. B.; Vailhen, D.; Amekraz, B.; Moulin, C.; Ezan, E.; Garin, J.; Bourguignon, J. The early response of Arabidopsis Thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Proteomics 2006, 6, 2180–2198. Stiborova, M. Cd2+ ions affect the quaternary structure of ribulose1,5-bisphosphate carboxylase from barley leaves. Biochem. Physiol. Pflanz. 1988, 183, 371–378. Dall’Osto, L.; Lico, C.; Alric, J.; Giuliano, G.; Havaux, M.; Bassi, R. Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light. BMC Plant Biol. 2006, 6, 32. Paddock, M. L.; Graige, M. S.; Feher, G.; Okamura, M. Y. Identification of the proton pathway in bacterial reaction centers: inhibition of proton transfer by binding of ZnCd2+ or Cd2+. Proc. Nat. Acad. Sci. U.S.A. 1999, 96, 6183–6188. Bandyopadhyay, D.; Chatterjee, A. K.; Datta, A. G. Effect of cadmium on purified hepatic flavokinase: involvement of reactiveSH group(s) in the inactivation of flavokinase by cadmium. Life Sci. 1997, 60, 1891–1903. Axelrod, H. L.; Abresch, E. C.; Paddock, M. L.; Okamura, M. Y.; Feher, G. Determination of the binding sites of the proton transfer inhibitors Cd2+ and Zn2+ in bacterial reaction centers. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1542–1547. Krupa, Z. Cadmium-induced changes in the composition and structure of the light-harvesting chlorophyll a/b protein complex II in radish cotyledons. Physiol. Plant 1987, 73, 518–524. Salt, D. E.; Smith, R. D.; Raskin, I. Phytoremediation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998, 49, 643–688. Bongi, G.; Loreto, F. Gas-exchange properties of salt-stressed olive (Olea europea L.) leaves. Plant Physiol. 1989, 90, 533–545.

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