Does the Different Proteomic Profile Found in Apical and Basal

Does the Different Proteomic Profile Found in Apical and Basal Leaves of Spinach Reveal a Strategy of This Plant toward Cadmium Pollution Response? Marco Fagioni and Lello Zolla* Department of Environmental Sciences, University of Tuscia, Viterbo, Italy Received December 29, 2008

Chlorosis develops in Spinacia oleracea L. plants exposed to Cd and is prevalently localized in the basal leaves. A proteomic comparison of basal and apical leaves from Cd-treated plants showed modified profiles that are different and complementary in the two locations. Total chlorophyll increased in apical leaves as did photosynthetic complexes and enzymes involved in CO2 fixation and carbohydrate metabolism. Thus, apical leaves seem to supply the plant’s energy requirements and, consistent with this, remain green after 40 days. In contrast, basal leaves experienced reduced chlorophyll a synthesis and photosynthesis, and later on an over production of ROS, which induces a cell defense response, leading to senescence and cell death. There was also an over production of GSH and phytochelatins, whose main role is in chelating Cd. These chelate-polypeptide complexes accumulate in the vacuole, limiting the distribution of Cd to apical leaves. In line, we found that many proteins involved in carbon metabolism were less abundant, whereas proteins involved in remobilizing carbon from other energy sources were up-regulated. We suggest that phytochelatin production has priority in Cd-stressed basal leaves and the nitrogen and sulfur metabolic pathways are activated for this purpose. Finally, as dead leaves detach from the plant, they carry away the sequestered Cd, thereby removing it completely from the plant and preventing any future access to the apical leaves. These events may represent an active detoxification strategy in higher plants. Keywords: plant response • Cd stress • 2D IEF-SDS-PAGE • phytochelatin • pigment analysis • Cd ion determination

Introduction Cadmium (Cd), a widespread pollutant, occurs in air and soils exposed to heavy traffic and industrial wastes. It is present in chemical fertilizers and is also released into the environment by power stations, heating systems, metal-working industries, waste incinerators, and cement factories. There is little evidence of it having any biological function1 though plenty to show that it induces multiple damages in every organism examined, including plants.2 In this respect, the main source of Cd exposure in the general population is through the food chain, and contaminated plants represent the first step. In plants, Cd stimulates the production of metabolic enzymes in the roots,3 while in leaves, the most obvious effect of Cd toxicity is chlorosis. Cadmium has a strong affinity for thiolates which renders it toxic to the plant, as important enzymes are inactivated when Cd binds to sulfydryl-groups (-SH).4 Moreover, due to the chemical similarity, Cd can replace Zn, Fe, and Ca in the prosthetic group of many proteins.5 Cd causes plant growth retardation and inhibition of diverse metabolic processes including photosynthesis, respiration and assimilation of nitrate. It also affects the accumulation of * To whom correspondence should be addressed. Prof. Lello Zolla, University of Tuscia, Largo dell’Universita` snc, 01100 Viterbo, Italy. Phone: 0039 0761 357100. Fax: 0039 0761 357179. E-mail: [email protected]. 10.1021/pr8011182 CCC: $40.75

 2009 American Chemical Society

various metabolites.6 With regard to the effects on photosynthesis, various studies have recorded stomatal closure,7 inhibition of carbon assimilation and photosynthetic electron transport have been demonstrated, often correlated with premature senescence of chloroplasts.8-10 Cd has been found to inhibit the synthesis of chlorophylls (Chl)11 and their stable binding to proteins,12 thereby decreasing the accumulation of pigmentlipoprotein complexes, particularly photosystem I (PSI).5,13,14 Over time, inhibition of photosynthesis induces oxidative stress,15 which can contribute to the degradation of photosynthetic structures and the induction of senescence,16,17 as well as stress acclimation of plants through signal-transduction processes.18 The proteomic profile of plants subjected to short exposures to Cd (several days) is well documented, while there is little information available for longer exposures. Briefly, at 24-48 h the main response is energetic. In Arabidopsis thaliana cell cultures exposed to different concentrations of Cd, proteomic analysis indicated that carbon, nitrogen and sulfur metabolic pathways were activated.19 Most notably, among the up regulated proteins were enzymes involved in the biosynthesis of glutamate, cysteine and glycine, which are all precursors in the formation of GSH and phytochelatins. Phytochelatins are enzymatically synthesized polypeptides with heavy metal-binding abilities; their general structure is (Glu-(Cys))nGly. Many different isoforms have been identified which are Journal of Proteome Research 2009, 8, 2519–2529 2519 Published on Web 03/16/2009

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species-specific. Phytochelatins and GSH can chelate Cd, and therefore detoxification of the cytosol is attained by accumulation of the chelate-polypeptide complexes in the vacuole.4 As phytochelatin and GSH synthesis represents an important sink for sulfur, this up-regulation explains the involvement of the sulfur metabolic pathway in Cd detoxification.4 Most investigations on Cd toxicity have considered leaf damage in the whole plant. This study, in contrast to poplar,20 takes into account the fact that Cd accumulates primarily in the basal leaves and does not reach the apical leaves until 40 days from first exposure, whereupon new leaves have matured. It is possible that this delay indicates the presence of an active plant defense mechanism. To investigate this possibility and throw some light on the nature of the mechanisms involved, we performed separate proteomic profiles of basal and apical leaves.

Materials and Methods Plant Material and Experimental Treatments. The spinach plants were 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).5 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 concentrations (20, 50, 100 µM) under continuous aeration at pH 5.5,21 while the other half were grown without Cd (control plants). Nutrient solution was changed every three days in both normal and Cd-treated plants. Twenty day old spinach plants (before beginning Cd treatment) had two cotyledon leaves plus six true leaves: the first four basal leaves (1-2 and 3-4) and the two apical ones (5-6). The two sets of basal leaves and apical leaves were harvested separately from plants after 0, 5, and 18 days of Cd treatment and analyzed separately for their biochemical composition and structure. Control samples were taken from untreated plants which were the same age as their treated counterparts. All the leaves were frozen with liquid nitrogen before being used for biochemical measurements. Protein Extraction and Two-Dimensional Electrophoresis. Leaves were finely ground in liquid nitrogen, and the protein extraction was done in agreeance with Hajheidari et al.22 The 2D IEF-SDS-PAGE was run in agree with D’Amici et al.23 Proteins were stained by Blue Sliver.24 Image Analysis. Two dimension gel images were digitized using an ImageScanner II (GE Healthcare, Uppsala, Sweden) with a resolution of 300 dpi and 16-bit greyscale pixel depth. Image analysis was carried out with Progenesis SameSpots software vers. 2.0 (Nonlinear Dynamics), which allows spots detection, quantification, and background subtraction. Each stage of leaves development (control, apical and basal Cdtreated leaves) 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.25 A match set was created from the protein patterns of five replicate gels for each independent leaf protein extract (at day 0, 5, and 18 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 2520

Journal of Proteome Research • Vol. 8, No. 5, 2009

Fagioni and Zolla volume of all spots on that image that are matched and presented in all of the gels of the experiment. Statistical analysis separated proteins that significantly increased or decreased after the treatments. A Student’s t test was performed to compare the two groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0.05. In Gel Digestion and Peptide Sequencing by Nano-RPHPLC-ESI-MS/MS. Bands and spots from 2D gels were carefully excised and subjected to in-gel trypsin digestion according to Shevchenko et al.26 Peptide mixtures were separated using a nano flow-HPLC system (Ultimate; Switchos; Famos; LC Packings, Amsterdam, The Netherlands) as previously done by Timperio.27 Protein identification was performed by searching in the National Center for Biotechnology Information non redundant (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). 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 as previously done by Timperio,27 using RPHPLC equipped with diode array detector for unambiguous identification of each xantophylls. 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.28 Phytochelatin Detection in Spinach Leaves Treated with Cadmium. The homogenate sample for PC analysis was prepared using the method of Grill29 and PCs were analyzed by precolumn derivatization using monobromobimane (mBBr). 400 mg of tissue was frozen in liquid nitrogen, pulverized and transferred to a microfuge tube. Then 0.4 mL of a freshly prepared solution of 1 N NaOH containing 1 mg of sodium borohydride (NaBH4) per ml was added. After thorough mixing the solution was centrifuged at 11.000g for 5 min at 4 °C. The supernatant was collected and acidified with 3.6 N HCl (ratio 5:1). The tubes were incubated in an ice bath for 15 min followed by centrifugation at 11.000g for 5 min at 4 °C. The 200 µL of this supernatant was diluted with 400 µL of 200 mM HEPES buffer (pH 8.2), then derivatized by adding 10 µL of 25 mM mBBr and incubated for 30 min in the dark. The reaction was stopped by adding 60 µL of 10 mM acetic acid. The final mixture was filtered through 0.45 µm filter and used for PCs analysis. Separation and analysis of PCs was carried out on reverse phase HPLC-ESI-MS (Perkin Helmer, Massachusetts) with AlltimaTM C18 (Alltech, Milan, Italy) with a guard column C18, using a gradient solution A and B (A containing 0.05% trifluoroacetic acid and B containing 26% acetonitrile in A solution) at a flow rate of 1.0 mL min-1. Fluorescence intensity with an excitation wavelength of 380 nm and an emission wavelength of 470 nm was recorded using a fluorescence detector (Perkin-Elmer, LC 240). The HPLC-ESI-MS experiments were performed using an ion trap Esquire 3000 plus in

Strategy of Spinacia oleracea L. in Response to Cd Stress

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positive ion mode (Bruker Daltonik, Bremen, Germany). The 1 mL min-1 flow of the analytical column was split post column, with 50 µL min-1 entering the mass spectrometer and 950 µL min-1 going to the fluorescence detector. For analysis with pneumatically assisted ESI, an electrospray voltage of 4.6 kV and a nitrogen sheath gas flow were employed. The temperature of the heated capillary was set to 300 °C. Phytochelatin mass spectra were recorded by scanning the first quadrupole, the scan range being 100-2000 amu. Cadmium 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).30 Statistical Analyses. Chl, pigment and phytochelatin 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 Turkey’s and Scheffe’s mean separation tests (R)0.05) for distinguishing among significantly deviating means. ROS Determination. Oxidized proteins were tagged with 2,4dinitrophenylhydrazine (DNPH) and detected as previously done by Levine.31

Results The investigation was carried out using 20 day old spinach plants, grown in hydroponic culture with Hoagland nutrient solution. These plants were treated with 100 µM Cd because when we exposed spinach plants to lower Cd concentrations (20, 50 µM), which would normally elicit a response in other species, they did not show significant changes. At this age, spinach plants have six leaves plus the two cotyledon leaves. The latter showed chlorosis after just 4-5 days and died after 9 days, while the first two basal leaves (leaves 1-2) showed a slight chlorosis after 6-7 days and died after 25 days. The other two basal leaves (leaves 3-4) first showed symptoms of chlorosis after 10-12 days and survived for over 30 days. Interestingly, the two apical leaves (leaves 5-6) did not show any chlorosis for a much longer time, up to about 40 days. Moreover, in treated plants, chlorosis started at the leaf margins and gradually spread toward the stem (see Figure 1A). There were no visual symptoms of pinpoint necrosis observed near the main leaf vein, as reported for youngest poplar leaves,4 but wide necrosis in the blade leaves. Figure 1B shows a representative plant at 15 days of treatment in which it can be seen evident that basal leaves 1 and 2 are dried, basal leaves 3 and 4 are visibly chlorotic, but no chlorosis is evident in the apical leaves, which appeared greener than controls even at 30 days (see Figure 1C). Interestingly, a further macroscopic observation was that plants growing in the presence of Cd had smaller leaves than the controls (see Figure 1C), indicating that Cd significantly reduces plant growth and leaf expansion. Samples were prepared from each pair of leaves, that is, the first two pairs of basal leaves (1-2 and 3-4) and the two apical ones (5-6), and these were analyzed separately for their biochemical and protein composition. Two new small leaves (7-8) appeared after 20-25 days of Cd treatment which were not used in our investigation. In accordance with observations at the macroscopic level, Cd was not detected in the apical leaves until 42 days, where it was revealed to be about 0.21 ( 0.01 nmol cm-2. In contrast, Cd concentration in the basal leaves ranged from 7.7 ( 0.1 (the first two basal leaves) to 3.9

Figure 1. Cd toxicity symptoms on Spinacia oleracea L. (A) Leaves showing Cd-induced chlorosis symptoms after 0, 5, 10, and 15 days of treatment. (B) Whole hydroponic plant showing the apical, basal leaves, and necrosis present after 15 days of Cd treatment. (C) Comparative picture showing the different growth rate of plants after 30 days of Cd treatment. On the left: control plant. On the right: Cd-treated plant.

( 0.1 (the second ones) nmol cm-2 after 10 days of Cd treatment, increasing over time. HPLC measurements were used to determine chlorophyll and xanthophyll concentrations in apical and basal leaves. Figure 2 shows a comparative chromatogram of pigments in basal leaves before and after adding Cd (15 days of treatment). The insert shows Chl a and b changes in both types of leaf. In the apical ones, a slight increase of both chlorophylls was observed with respect to control leaves, while in the basal leaves, both chl a and b decreased just after the first 5 days of treatment and continued to decrease over the course of the experiment. It was evident that [Chla] decreased significantly more than [Chlb], especially during the first 10 days of treatment. A different pattern was observed for the xanthophyll pigments; in basal leaves some xanthophylls (lutein, neoxanthin and violaxanthin) increased throughout the Cd treatment. The effect was particularly evident for lutein in leaves treated with Cd for 15 days (see Figure 2) whereas in the apical leaves there were no significant xanthophyll changes (data not shown). Proteomic Investigations. We used IEF-PAGE 2D electrophoresis to compare the protein profile time courses in apical and basal leaves of Cd treated plants with those from controls. Journal of Proteome Research • Vol. 8, No. 5, 2009 2521

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Figure 2. RP-HPLC pigment chromatograms, showing Lutein (L), Neoxanthin (N), Violaxanthin (V), Chlorophyll b (Chl b), and Chlorophyll a (Chl a). (A) Overlaid RP-HPLC chromatograms of pigments extracted from 0 days Cd-treated apical leaves (gray line) and from 18 days Cd-treated apical leaves (black line). (B) Overlaid RP-HPLC chromatograms of pigments extracted from 0 days Cd-treated basal leaves (gray line) and from 18 days Cdtreated basal leaves (black line). The inserts show the time-course of leaf chlorophylls production during the treatment.

Basal and apical leaves were harvested separately from plants after 0, 5, and 18 days of Cd treatment. Ten leaves were collected from ten different plants of the same age, but grown in different periods in order to consider the influence of any biological variation. Likewise, on the same days basal and apical leaves were also harvested from the control plants. Each stage of leaf development (both of control and Cd-treated plants) was analyzed in 5 replicates. Proteomic Investigations of Apical Leaves. Figure 3A shows the master map obtained by comparing maps at 0, 5, and 18 days of Cd treatment. It can be seen that although Cd had not reached the apical leaves after 18 days significant changes were observed in the proteomic profile. No new proteins were observed but up regulated ones. The protein(s) contained in each spot were identified by in gel trypsin digestion and by tandem mass spectrometry. All proteins identified with this procedure are listed in Table 1. Moreover, Supplemental Table 1S with peptide sequences is provided in the Supporting Information. Figure 3B shows histograms of modulated spots, obtained after image analysis with Progenesis SameSpot software (NonLinear Dinamics, New Castle, UK) and the relative protein identification. It can be observed that most of the differential proteins are up regulated and are involved in photosynthesis, such as PSI and PSII antenna, correlating with Chl increase, ATP synthase and NADH dehydrogenase. Proteins involved in carbohydrate metabolism and CO2 fixation were also more abundant, such as rubisco, carbonic anhydrase, triosephosphate isomerase and fructose-bisphosphate aldolase. 2522


Fagioni and Zolla

Figure 3. Images of “Blue Silver” stained 2D IEF-SDS PAGE gels. (A) Image of “master map” representing the summarizing map of the three samples after image analysis. Each spot differently expressed is indicated by its relative number. (B) Histogram of modulated spots obtained after image analysis of Cd-treated apical leaves 2D IEF-SDS-PAGE. Each spot is shown regarding its expression during the Cd treatment. The box 1 represents the Cd-treated apical leaves at 0 day of treatment, the box 2 and 3 one represent the Cd-treated apical leaves at 5 days and 18 days of Cd treatement, respectively.

Enzymes involved in Rubisco biosynthesis, such as chaperonin cpn21, and in O2 production, like oxygen evolving complex 1 and 2, are also up regulated. Proteomic Investigations of Basal Leaves. Figure 4A shows the master map obtained by comparing maps at 0, 5, and 18 days of Cd treatment. The protein(s) contained in each spot were identified by in gel trypsin digestion and by tandem mass spectrometry. All proteins identified with this procedure are listed in Table 2. Moreover, Supplemental Table 2S with peptide sequences is provided in the Supporting Information. Figure 4B shows histograms of modulated spots, obtained after image analysis with Progenesis SameSpot software (NonLinear Dinamics, New Castle, UK) and the relative protein identification. It can observed that most of the proteins that showed a significant up-regulation are involved in the oxidative stress response, carbohydrate metabolism, pathogen regulated protein and cysteine biosynthesis, while down regulated proteins are involved in photosynthesis, carbohydrate metabolism, O2 production, and protein folding. Another interesting point is that most of those proteins that are up regulated in apical leaves were down regulated in basal

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Strategy of Spinacia oleracea L. in Response to Cd Stress Table 1. List of Protein Identified by MS in Cd-Treated Apical Leaves spot no

2 7 23 47 52 55 59 63

4 5 8 10 12 15 17 20 24 25 29 31 33 36 67 51 68

NCBI accession number

protein name

no of identified peptides

Mascot score

fold of variation (5 d/18 d)a

33 15

1130 599

+0.31/+0.54 +0.27/+0.13

2 11 7 1 18 18

184 508 337 73 531 531

+0.88/+0.96 +0.73/+0.58 +0.64/+0.61 +0.62/+0.44 +0.42/+0.52 +0.37/+0.43

gi|115453971 gi|133917261 gi|126022791 gi|133917261 gi|115774

31 11 6 16 2

436 238 361 400 90

+1.27/+1.34 +1.21/+1.35 +1.07/1.21 +1.12/+1.17 +1.13/+1.15

gi|19184 gi|126022791 gi|115453971 gi|217071344 gi|126022791 gi|126022791 gi|115802

3 24 15 13 36 23 4

228 689 314 291 911 732 150

+1.11/+0.92 +1.05/+0.92 +0.97/+1.01 +0.98/+0.98 +0.96/+0.78 +0.92/+0.70 +0.90/+0.75

gi|430947 gi|115780

9 2

210 122

+1.38/+0.74 +0.79/+0.70

gi|3764067

2

79

+0.40/+0.36

3

183

+0.64/+0.58

56

815

+0.33/ +0.24

3 9

141 449

+0.70/+0.46 +0.45/+0.48

Carbohydrate metabolism Rubisco large subunit gi|11497536 Chain L, Activated Spinach Rubisco In Complex With The gi|2392029 Product 3- Phosphoglycerate Triosephosphate isomerase, chloroplast precursor (TIM) gi|1351271 fructose-bisphosphate aldolase gi|22633 fructose-bisphosphate aldolase gi|22633 Carbonate anhydrase gi|18401719 Rubisco large subunit gi|11497536 Chain A, Activated Spinach Rubisco Complexed With 2gi|1827835 Carboxyarabinitol Bisphosphate Photosyntesis Os03g0592500 major chlorophyll a/b binding protein LHCb1.2 ATP synthase CF1 alpha subunit major chlorophyll a/b binding protein LHCb1.2 Chlorophyll a-b binding protein 13, chloroplast precursor (LHCII type I CAB-13) (LHCP) Type I (26 kD) CP29 polypeptide ATP synthase CF1 alpha subunit Os03g0592500 Chlorophyll A-B binding protein ATP synthase CF1 alpha subunit ATP synthase CF1 alpha subunit Chlorophyll a-b binding protein 36, chloroplast precursor (LHCII type I CAB-36) (LHCP) PSI type III chlorophyll a/b-binding protein Chlorophyll a-b binding protein, chloroplast precursor (LHCII type I CAB) (LHCP) NADH deidrogenase

Oxygen production 33 kDa precursor protein associated with the photosynthetic gi|131385 oxygen-evolving complex from spinach photosystem II oxygen-evolving complex protein 1 precursor gi|21283 spinach Protein folding

45 61

chloroplast chaperonin 21 chloroplast elongation factor TuB (EF-TuB)

gi|50660329 gi|218312

a Fold of protein variation is calculated by taking the mean of the normalized volume of the sample at 0 days of treatment and comparing it with the mean of the normalized volume of the sample at 5 days and 18 days of treatment, respectively. The mean of normalized volume was calculated by Progenesis SameSpots image analysis software.

leaves. This suggests that apical and basal leaves react differently to Cd treatment probably because they perform different roles in the plant’s survival response to this toxic metal.

protein fragmentation could all be caused by active oxygen species, generated during senescence, which randomly attack proteins.32

A further observation that deserves attention was revealed through IEF-PAGE 2D electrophoresis of basal leaves at 42 days of Cd treatment (data not shown) where faint spots could be seen in the central part of the gel that were not present in the control, and a higher number of new spots showed up in the low molecular masses region. Concomitantly, high-molecularmass aggregates, seen as a smearing in the upper part of the gel, appeared in the region around 300 kDa. Decreased intensity of staining with Coomassie blue and an increase in the number of low molecular weight spots in SDS-PAGE are two phenomena that are usually observed when proteins are exposed to active oxygen species, suggesting that most lesions are due to oxidative processes. In fact, the presence of smearing, mobility shift of intrinsic protein bands, aggregate formation, and also

Carbonylated Protein As Biomarker of ROS Generation. The presence of protein oxidation inside leaves was measured as total carbonyl group content by reaction with 2,4-dinitrophenylhydrazine (2,4-DNPH), on the assumption that carbonyl proteins are a marker for ROS generation.31 Carbonylated proteins appeared after just 5 days of Cd treatment in the basal leaves and increased over time, while they were not detected in the apical leaves (data not shown). Detection of Phytochelatins. Phytochelatins (PCs) were separated and identified by HPLC coupled on line with a mass spectrometer. Samples from basal leaves after 0, 10, and 15 days of Cd treatment were injected onto a C18 column for chromatographic separation. Table 3 reports the identified phytochelatin structure, the molecular weight, the detected m/z after derivatization with mBBr, and the relative retention time. Journal of Proteome Research • Vol. 8, No. 5, 2009 2523

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Figure 4. Images of “Blue Silver” stained 2D IEF-SDS PAGE gels. (A) Image of “master map” representing the summarizing map of the three samples after image analysis. Each spot differently expressed is indicated by its relative number. (B) Histogram of modulated spots obtained after image analysis of Cd-treated apical leaves 2D IEF-SDS-PAGE. Each spot is shown regarding its expression during the Cd treatment. Box 1 represents the Cdtreated basal leaves at 0 day of treatment, boxes 2 and 3 represent the Cd-treated basal leaves at 5 days and 18 days of Cd treatment, respectively. Histogram of each spot showing the up or down regulated proteins of Cd-treated apical leaves and the relative protein identification.

In the control leaves, small amounts of PC2, PC3, PC4, desGLU(PC3), and desGLU(PC4) were present, but these increased significantly in Cd treated leaves after 15 days, by 3, 1.5, 7.5, 2, and 2 fold, respectively (Figure 5A). New phytochelatins also appeared in Cd-treated leaves, which were identified as homo PC2, homo PC3, isoPC3(ser), isoPC4(ser), PC4, and desGlu PC4. The semiquantitative analysis of changes in phytochelatin concentration in leaves during the Cd treatment is shown in Figure 5B. We repeated the same experiment in apical leaves after 15 days of Cd treatment. Phytochelatin concentration was found to be no different from that of control leaves, suggesting that Cd was not present in these leaves and hence no damage had occurred there (data not shown).

Discussion Spinach is unusually resistant to high concentrations of cadmium and, as such, provides a good model system for 2524


Fagioni and Zolla evaluating the effects of prolonged Cd exposure.5 This is of interest because most investigations have involved short exposures and little information exists on how plants respond to longer exposures. We report here that Cd gradually accumulates in the basal leaves working its way up the plant and eventually reaching the apical leaves, though only at a much later time (40-42 days). That Cd is prevalently localized into the basal leaves, although the branching points of the basal and apical leaves are very close to each other, is related to the mobility of the ion inside plants. It allows the Cd to enter first into the two cotyledon leaves, which die first and subsequently into the two first true leaves (1 and 2) and so on. Cd only enters the apical leaves after about 40 days, as revealed by atom absorption and by the evidently green leaves. Clearly phytochelatins play a crucial role in delaying this migration, allowing the intracellular chelation of Cd and its compartmentalization within the vacuole. This limits the amount of free Cd that can circulate to the upper leaves and may represent a detoxification strategy adopted by higher plants, possibly as part of a general mechanism that is also implemented to exclude toxic anions such as Cl-. The appearance of chlorosis is a symptom that phytochelatins are fully saturated and free Cd then substitutes bivalent ions inside the proteins and produces ROS. This is due to the fact that the Cd concentration is higher than that of the phytochelatins (100 µM of Cd was intentionally chosen) therefore the surplus Cd substitutes the bivalent ions of many proteins as well as causing the production of ROS and appearance of chlorotic symptoms. Regarding the visible signs of chlorosis which start from the leaf edge and eventually spread toward the stem, this is related to the process of cell expansion in the leaf edge, and this part is more sensitive to Cd, as revealed in Thlapsi caerulescens by X-ray microanalysis.35 After longer time periods, chlorotic leaves dry and detach from the plant, carrying away the sequestered Cd and thereby removing it completely from the plant and preventing any future access to the apical leaves. Apical leaves remain efficient for more than 40 days, with a higher rate of photosynthesis than normally observed and which provides all the energy needs of the rest of plant. Thus plants seem to be divided into two different parts having different and complementary functions. This is reflected by the two different and complementary proteomic profiles found in basal and apical leaves. Cd reduces the leaf growth of all plants. Leaves of Cd treated planted plants are about 60% smaller than those of controls of the same age. In a previous paper, plants exposed to 100 µM Cd showed an increase in cytokinin oxidase, an enzyme induced by the increase in ABA concentration, which typically occurs under environmental stresses, causing fewer cell divisions and therefore impairing normal development.36 However, it is important to underline that chlorosis resulting from Cd exposure is not the same phenomenon as that observed in Fe deficiency, where both apical and basal spinach leaves showed chlorosis after just 4-5 days.27 Interestingly, the symptoms of toxicity seemed to be less severe in spinach plants than in poplars as the former developed large necrotic spots, whereas the latter had small necrotic spots mostly near the main leaf veins.4 Moreover, in Poplar Cd accumulated at a faster rate in stems followed by a linear increase in the leaves, though this remained lower than in stems. This illustrates the genetic variability which has been found between species and within the same species in response to Cd exposure, for example pea

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Strategy of Spinacia oleracea L. in Response to Cd Stress Table 2. List of Proteins Identified by MS in Cd-Treated Basal Leaves spot no

23 27 36 78 26 27 33 34 41 44 45 48 56 63 64 80 85

NCBI accession number

protein name

Superoxide dismutase [Cu-Zn] arginase Superoxide dismutase [Cu-Zn] thioredoxin peroxidase

Response to oxidative stress gi|134686 gi|15236640 gi|134628 gi|18654477

no of identified peptides

Mascot score

fold of variation (5 d/18 d)a

4 14 5 7

262 436 147 63

+0.27/+0.83 -0.72/+0.10 -0.02/+0.42 +0.10/+0.35

13 3 4

534 224 187

-0.31/-0.42 -0.72/+0.10 -0.22/-0.59

48 26

1401 622

+0.02/-0.41 -0.22/- 0.63

45

1477

-0.37/-0.47

36

977

-0.17/-0.53

50 16

1704 714

-0.29/-0.58 -0.35/-0.37

29 91 39

845 1745 1522

-0.09/-0.37 -0.07/-0.39 -0.07/-0.25

44

550

-0.05/-0.25

2 3 1 4

83 166 70 242

-0.08/-0.51 -0.14/-0.41 +0.52/+0.51 -0.03/-0.32

9 2

184 75

+0.27/+0.83 -0.05/-0.29

6

389

+0.31/-0.42

14

545

+0.02/+0.41

gi|1015370 gi|21309

9 27

124 448

-0.53/-0.73 -0.52/-0.68 -0.03/-0.29

Chlorophyll biosynthesis gi|19875 gi|3334150

8 2

359 64

-0.31/-0.42 -0.35/-0.43

31 4 11 27

836 165 364 782

-0.09/-0.65 -0.12/-0.37 -0.09/-0.39 -0.05/-0.30

Carbohydrate metabolism rubisco activase precursor gi|170129 malate dehydrogenase gi|10798652 Phosphoribulokinase, chloroplast precursor (PRKase) (PRK) gi|125579 (Phosphopentokinase) rubisco activase precursor gi|170129 Ribulose bisphosphate carboxylase large chain precursor (RuBisCO gi|2500656 large subunit) Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast gi|12643998 precursor (RuBisCO activase) (RA) Ribulose bisphosphate carboxylase large chain precursor (RuBisCO gi|132051 large subunit) rubisco activase precursor gi|170129 RuBisCO large subunit-binding protein subunit alpha (60 kDa gi|3790441 chaperonin subunit alpha) (CPN-60 alpha) chloroplast ribose-5-phosphate isomerase gi|18654317 rubisco activase precursor gi|170129 Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast gi|12643998 precursor (RuBisCO activase) ribulose 1,5-bisphosphate carboxylase small subunit gi|2529378 Photosynthesis

31 43 47 73

chlorophyll a/b-binding protein type III Plastocyanin chloroplast precursor Photosystem I reaction center subunit IV, chloroplast precursor (PSI-E) ATP synthase delta chain, chloroplast precursor

23 76

outer membrane lipoprotein-like major latex like protein homologue

gi|7271947 gi|130285 gi|131178 gi|114584

Pathogen related protein gi|21553811 gi|14594813

50

Cysteine biosynthesis S-adenosylmethionine synthetase 2 (AdoMet synthetase 2) (Methionine gi|127046 adenosyltransferase 2) (MAT 2) cysteine synthase gi|303902

28 35 75

24 kDa RNA binding protein 28kD RNA binding protein 13.3 kDa protein P16.5

26 49

glutamate-1-semialdehyde 2,1-aminomutase Magnesium chelatase

30 51 67 77

Plastid-specific 30S ribosomal protein 2 Low PSII Accumulation1 50S ribosomal protein L12 chloroplast mRNA-binding protein CSP41 precursor

26

Protein folding

Protein synthesis gi|75275079 gi|18405391 gi|133085 gi|1532135

a Fold of protein variation is calculated by taking the mean of the normalized volume of the sample at 0 days of treatment and comparing it with the mean of the normalized volume of the sample at 5 days and 18 days of treatment, respectively. The mean of normalized volume was calculated by Progenesis SameSpots image analysis software.

genotypes.37 Each plant is differently damaged depending on how Cd is translocated, distributed, and consequently accumulated. Cd Ions Only Reach Apical Leaves after Longer Exposure Times. It is interesting that in spinach and in brassica,5 and presumably in many other species, the apical leaves are not reached by Cd ions, unless exposure is prolonged. In the case of spinach this occurs after 40 days, when new leaves have grown. Apical leaves remained green and no visual symptoms

were observed, despite a Cd concentration of 100 µM. Cd measurements as well as chlorophyll determination, showed the absence of Cd inside these apical leaves and Chl pigment was slightly increased with respect to the control. Consistent with this, some proteins involved in photosynthesis were found to be up regulated as well as proteins involved in CO2 fixation, such as Rubisco and carbonic anhydrase. In agreement, proteins involved in O2 production, such as OEC1 and OEC2, Journal of Proteome Research • Vol. 8, No. 5, 2009 2525

research articles

Fagioni and Zolla

Table 3. PCs, Homo-PCs, and Iso-PCs Characterized in Spinacia oleracea Plant Extract

a

peak

compound

molecular weight

m/z +mBBra

retention time

1 2 3 4 5 6 7 8 9 10 11 12

PC2: (γGlu-Cys)2-Gly DesGluPC4: Cys-(γGlu-Cys)4-Gly PC3: (γGlu-Cys)3-Gly DesGluPC3: Cys-(γGlu-Cys)2-Gly PC4: (γGlu-Cys)4-Gly Homo-PC4 (β-Ala): (γGlu-Cys)4-Ala Iso-PC2(Glu): (γGlu-Cys)2-Glu Iso-PC3 (Gln): (γGlu-Cys)3-Gln Homo-PC2 (β-Ala): (γGlu-Cys)2-Ala Homo-PC3 (β-Ala): (γGlu-Cys)3-Ala Iso-PC4 (Ser): (γGlu-Cys)4-Ser Iso-PC3 (Ser): (γGlu-Cys)3-Ser

540.60 874.98 772.92 642.72 1003.11 1018.14 611.65 842.93 553.61 785.88 1034.13 801.87

920.41 1635.84 1341.63 1213.63 1764.84 1778.84 992.42 1413.63 934.42 1356.63 1794.84 1372.63

12.35 18.60 23.77 24.41 31.06 11.95 15.44 16.47 19.53 19.97 35.94 36.10

Electrospray positive ionization mode.

Figure 5. Comparative phytochelatin RP-HPLC chromatograms. (A) HPLC profiles of mBBr derivatizated crude extract from basal leaves of cadmium-treated spinach plants at 0 and 15 days. Peaks denote the following phytochelatins: 1, PC2, 2, DesGluPC4, 3, PC3, 4, DesGluPC3, 5, PC4, 6, Homo-PC4(β-Ala), 7, Iso-PC2(Glu), 8, Iso-PC3 (Gln), 9, Homo-PC2(β-Ala), 10, Homo-PC3(β-Ala), 11, Iso-PC4(Ser), 12, Iso-PC3(Ser). The peak of the mBBr is also indicated. All the values are the mean of the triplicates ( SD. (B) Level of PCs, homo-PCs, and iso-PCs recorded in basal leaves of cadmium-treated plants at 0, 10, and 15 days. All the values are mean of triplicates ( SD. ANOVA significant at p e 0.01.

were up regulated. Moreover, to provide the necessary energy to the rest of the plant, glycolysis enzymes were also up regulated, indicating increased biological activity in these cells. Basal Leaves Are the Most Affected Leaves. Photosynthesis Damage. In the basal leaves, a significant down regula2526


tion of both magnesium chelatase and glutamate-1-semialdehyde 2,1-aminomutase was recorded. The former enzyme performs the first committed step in chlorophyll and (bacterio)chlorophyll biosynthesis with the insertion of Mg2+ into protoporphyrin IX,38 whereas the second one is a key enzyme in chlorophyll biosynthesis.4 Thus it is not surprising that chlorophyll a concentration was found to decrease during Cd treatment, being the first chl to be synthesized and subsequently biotransformed into chl b. The specific Chl a reduction consequently induces a general progressive decrease of PSI antenna, which contain more chl a than chl b.39 PSI breakdown is associated with the disappearance of major proteic supercomplexes. In our previous work, we reported that Cd triggers the accumulation of “incomplete” PSI which in turn is associated with an increase of the protein Ferrodoxin NADP oxidoreductase (FNR).5 This leads to an over production of H+, ATP, and NADPH+, all of which are necessary for the production of phytochelatins (PCs). Consistent with this data, our investigation revealed an increase in PSI-E. This protein stabilizes the interaction between psaC and the PSI core, assisting the docking of ferredoxin to PSI and interacts with FNR.40 Moreover, in the present study a decrease of Plastocyanin (Pc) was found. It is a small blue copper containing protein (10.5 kDa) found in the thylakoid lumen of the chloroplast which functions as an electron carrier protein between Cytochrome b6f and Photosystem I. Levels of Cytochrome b6/f and the ATP-synthase complex, which do not contain chlorophyll pigments, remained the same during the first days of Cd treatment,5 but a reduction in the amount of the ATP synthase delta subunit, which belongs to the ATPase delta chain family, was recorded over time. Contrary to what observed for PSI, no inhibition of a specific PSII protein was observed during the first 9-15 days, beside a significant accumulation of the “monomeric form CP43 less”.5 In agreement, in the present investigation a down expression of Low PSII accumulation 1 (LPA1) protein was recorded. LPA1 appears to be an integral membrane chaperone that is required for an efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1 during abiotic stress.41 Down Regulation of Protein Involved in Energetic Metabolic Pathways. The down regulated proteins involved in carbohydrate metabolism included Ribulose bisphosphate carboxylase/oxygenase activase, (RuBisCO activase), ribulose 1,5bisphosphate carboxylase small subunit (Rubisco small subunit), with a consequent lowering of the efficiency of CO2 fixation.4

Strategy of Spinacia oleracea L. in Response to Cd Stress In agreement, in poplars the carbon fixation and Calvin cycle showed also numerous key proteins less abundant in cadmium conditions, including RuBisCO activases, 60 kDa chaperonines, Ribulose-phosphate 3-epimerase, and aldolases.20 Similarly all these enzymes were found to be down regulated in rice leaves after Cd treatment and in many other organisms.42 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.43 In Cd-treated cyanobacterial spheroplasts, Rubisco activity also decreased to 40% of the control level, and in Cd-treated spinach chloroplasts Rubisco dropped to only 2.5% of the control level, demonstrating that Cd severely compromises Rubisco in higher plants.44 Beside the carbon fixing enzymes, there were reduced amounts of other proteins involved in carbohydrate metabolism, such as phosphoribulokinase, phosphoglycerate kinase and ribose 5-phosphate isomerase (RPI). Phosphoribulokinase is an enzyme involved in acquisition and assimilation of inorganic carbon, and this has also been shown to decrease during stress in a study that examined gene expression.45 The phosphoglycerate kinase is a key enzyme in glycolisis and catalyzes the formation of 3-phosphoglycerate from 1,3-bisphosphoglycerate. It is a further evidence that glycolitic pathways is severe damaged in Cd treated basal leaves. The down regulation of RPI, a key enzyme in the Calvin Benson Cycle, further indicates that in basal leaves the defense system is activated only against Cd and not for energy production. Moreover, while at short exposure time the main response of the vegetable cell to the Cd stress was the activation of nitrogen and sulfur pathways, in an effort to sustain the cells in terms of energy demand,19 after longer exposure these enzymes tuned to be out down regulated. Interestingly, MDH is up regulated. It catalyzes the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate in carbohydrate metabolism, with an overproduction of NADH, need for the PCs synthesis. Phytochelatins Overproduction. In basal leaves, we observed a rapid increase in existing phytocheatins and the appearance of new phytochelatins, which was similar to observations made in Arabidopis cells.19 Phytocheatins are small peptides containing cysteine, glutamate and glycine, which rapidly become the most abundant class of thiol compounds in the root cells. The Cd induced burst of PC biosynthesis is usually accompanied by a transient depletion of cell GSH pools that may be perceived by plants as indicating an additional need for sulfur.46 The role of sulfur amino containing amino acid metabolism in plant stress-resistance mechanisms is further explored in relation to the participation of cysteine as a precursor of peptides (gluthatione and phytochelatins) that chelate heavy metals47 and facilitate detoxification of active oxygen species.48 In fact, increased cysteine biosynthesis could also be an attempt to limit oxidative stress and ROS propagation.4 Consistent with this, the basal leaves in Cd treated spinach plants, up regulate cysteine synthase and S-adenosylmethionine synthetase 2, both involved in cysteine biosynthesis.49 However, although the distribution of the various classes of iso-PCs are presumed to differ from one plant species to another, our metabolite profiling analyses demonstrate that spinach cells are able to synthesize nearly all the families of PCs or PC-related peptides which have been described in

research articles different plant species. As a consequence, these results reinforce the hypothesis that it is the presence of the substrate (i.e., GSH isoforms), and not the specificity of the enzyme, that determines the nature of the PCs synthesized.19 Senescence. In basal leaves, among those proteins up regulated were several proteins from the oxidative stress response mechanism. The presence of Cd probably created a cellular condition of oxidative stress and ROS overproduction, resulting in the activation of general stress response proteins, like Pathogen Related protein(s) (PR), normally associated with the oxidative burst during biotic stress responses.50 Outer membrane lipoprotein-like proteins or lipocalins also increased. They have been implicated in many important functions, such as modulation of cell growth and metabolism, binding of cell-surface receptors, membrane biogenesis and repair, induction of apoptosis, animal behavior, and environmental stress response.51 This idea of oxidative stress is supported by direct measurement of ROS and by the appearance of visual symptoms, like necrotic spots and a substantial reduction in growth. Furthermore, proteinases, generally expressed during PCD and in senescent tissues, were increased. It has already been show that the production of reactive oxygen species (ROS) is one of the major plant responses to the presence of Cd ions.52 In our investigation, in fact, over production of ROS was evident after just 5 days of Cd treatment and continued to increase over time. An isoform of thioredoxin peroxidase was also found to have increased in abundance. Thioredoxin is very active in antioxidant defense and is regulated by endogenous and environmental stimuli at both the transcript and protein levels.53 The three isoforms of class III peroxidases were also up-regulated as part of the earliest response to Cd in Arabidopsis thaliana and Arabidopsis halleri,54 indicating that the up-regulation of sulfate activation is indeed the most pronounced and robust response to Cd induced consumption of reduced sulfur. Since ROS oxidize all sorts of cellular components, it was no surprise to find that arginase had been up regulated. This enzyme inhibits the production of nitric oxide (NO) via several possible mechanisms, including competition with NO synthase (NOS) for the substrate L-arginine.55 Arginase also catalyzes the formation of proline, an amino acids with apparently numerous and diverse roles under osmotic stress conditions, such as stabilization of proteins, membranes and subcellular structures as well as protecting cellular functions by scavenging reactive oxygen species.56 When reactive species such as ROS and RNS are over produced, the plant can protect itself by reducing all other activities, including protein biosynthesis. So it was consistent that chloroplast mRNA-binding protein CSP41, 24 kDa RNA binding protein, 28 kDa RNA binding protein, Plastid-specific 30S, and the 50S ribosomal L12 protein were all found to be down regulated. Finally, we noticed that over a longer exposure time (about 40 days) protein fragments started to appear in the low molecular weight region of the gels which are due to the increased proteolytic activity of denatured proteins, a phenomena previously observed in the Cd-treated Arabidopsis19 and common when ROS are over produced.21 In line, the presence of 20S proteasome is in agreement with findings reported by Kieffer4 who showed that over-expression of the a-subunit of the 20S proteasome, which conferred resistance to nickel, Cd, and cobalt in maize. Journal of Proteome Research • Vol. 8, No. 5, 2009 2527

research articles Conclusion A question that comes up time and again in stress physiology is which of the changes detected under certain conditions are specific responses to the stimulus under investigation and which ones are secondary responses to damage. Clearly, during senescence the borderline is lost. However, taking into account all the data from this study, a picture begins to emerge of what may happen in spinach leaves during Cd exposure. Cadmium does not reach all leaves simultaneously but appears to infiltrate the lower leaves first, these then overproduce phytochelatins in an attempt to prevent Cd spreading to younger leaves. Free Cd is able to substitute cationic ions inside proteins5 and produce ROS, pushing affected leaves into irreversible senescence. If Cd is present in excess it can reach the upper leaves, however until that happens the apical leaves are induced to overexpress photosynthetic enzymes to provide sufficient energy to supply the rest of the plant. As dead leaves detach from the plant, they carry away the sequestered Cd, thereby removing it completely from the plant and preventing any future access to the apical leaves. Although the original apical leaves are also reached by a small amount of Cd new upper leaves continue to grow, presumably allowing the plant to out run its adversary as long as the toxic exposure remains finite. Abbreviations: Cd, cadmium; ROS, reactive oxygen species; GSH, gluthatione; PS, photosystem; Chl, chlorophyll; IEF, isoelectro focusing; SDS, sodium dodecyl phosphate, PC, phytochelatin; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; FA, formic acid; PAGE, polyacrylamide gel electrophoresis; PS, photosystem; RP-HPLC, reversed-phasehigh performance liquid chromatography.

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Does the Different Proteomic Profile Found in Apical and Basal

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