Journal of Experimental Botany, Vol. 52, No. 354, pp. 25-35,
January 2001
© 2001 Oxford University Press
Original Papers |
Iron deficiency differently affects peroxidase isoforms in sunflower
1 Dipartimento Chimica e Biotecnologie Agrarie, Università degli Studi di Pisa, Via del Borghetto 80, 56124 Pisa, Italy
2 Dipartimento Biologia, Università degli Studi di Padova, Via Trieste 75, 35121 Padova, Italy
Received 23 June 2000; Accepted 26 July 2000
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Abstract |
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The response of both specific (ascorbate peroxidase, APX) and unspecific (POD) peroxidases and H2O2 content of sunflower plants (Helianthus annuus L. cv. Hor) grown hydroponically with (C) or without (-Fe) iron in the nutrient solution were analysed to verify whether iron deficiency led to cell oxidative status. In -Fe leaves a significant increase of H2O2 content was detected, a result confirmed by electron microscopy analysis. As regards extracellular peroxidases, while APX activity significantly decreased, no change was observed in either soluble guaiacol or syringaldazine-dependent POD activity following iron starvation. Moreover, guaiacol-dependent POD activity was found to decrease in both ionically and covalently-cell-wall bound fractions, while syringaldazine-POD activity decreased only in the covalently-bound fraction. At the intracellular level both guaiacol-POD and APX activities underwent a significant decrease. The overall reduction of peroxidase activity was confirmed by the electrophoretic separation of POD isoforms and, at the extracellular level, by cytochemical localization of peroxidases by diaminobenzidine staining. The electrophoretic separation, besides quantitative differences, also revealed quantitative changes, particularly evident for ionically and covalently-bound fractions. Therefore, in sunflower plants, iron deficiency seems to affect the different peroxidase isoenzymes to different extents and to induce a secondary oxidative stress, as indicated by the increased levels of H2O2. However, owing to the almost completely lack of catalytic iron capable of triggering the Fenton reaction, iron-deficient sunflower plants are probably still sufficiently protected against oxidative stress.
Key words: H2O2, iron deficiency, oxidative status, peroxidase, sunflower.
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Introduction |
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Adverse environmental growth conditions are reported to induce degradative processes in plants as a consequence of a generation of partially reduced oxygen species (activated oxygen). It is now well documented that pathogen attack (Levine et al., 1994
; Vanacker et al., 1998), atmospheric pollution (Ranieri et al., 1996, 1998, 1999a) and drought conditions (Moran et al., 1994) lead to perturbation of the redox state towards an oxidative metabolism within plant cells.
With regard to environmental perturbations such as nutrient deficiency, evidence has been reported, both in Mg-deficient bean leaves and Mn-deficient needles of Norway spruce trees, of the increase in activity of several antioxidant enzymes (Cakmak and Marschner, 1992; Polle et al., 1992). On the other hand, there is little information about the relationship between iron-deficiency and the onset of oxidative stress status (Iturbe-Ormaetxe et al., 1995) despite both the world-wide problems represented by scarce iron bioavailability in the soil and the well-known double role which iron plays within cell metabolism. In fact, iron is either a constituent or a cofactor of many antioxidant enzymes, and can act as a pro-oxidant factor because free or loosely bound it catalyses free radical generation in the presence of reductants and peroxides through the Fenton reaction. In particular, Fe is involved in the Fe-catalysed Haber–Weiss reaction in which trace amounts of Fe3+ are reduced by 
to produce Fe2+ which, in turn, reacts with H2O2 to form OH. (Fenton reaction).
As the intrinsic constituent or metal cofactor, iron is actively involved in cellular detoxification reactions catalysed by catalase (CAT, EC 1.11.1.6), phenolic-dependent peroxidases (non-specific peroxidases, POD, EC 1.11.1.7), ascorbate peroxidases (APX, EC 1.11.1.11) and Fe superoxide dismutase (Fe-SOD, EC 1.15.1.1), which scavenge hydrogen peroxide and superoxide, thus protecting the cell from oxidative injury.
Peroxidases are a large family of ubiquitous enzymes which contain iron as heme and are responsible for both the scavenging of H2O2 by the oxidation of phenols, and its generation, through the oxidation of NADH (Halliwell, 1978; Otter and Polle, 1997; Polle et al., 1994). Peroxidases which use ascorbate as a reductant (APX) are specifically involved in the H2O2 detoxification in chloroplasts (Asada, 1992) and the cytosol (Mittler and Zilinskas, 1991). Recently, many studies have also reported the presence of APX in the apoplast environment (Castillo and Greppin, 1986, 1988; Ranieri et al., 1996, 1998, 2000; Vanacker et al., 1998). By contrast, different functions have been reported to be carried out by ‘unspecific’ PODs by catalysing the oxidation of a wide range of phenolic substrates (Siegel, 1993). Individual POD isoenzymes are present in numerous cell compartments, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, cytosol, the vacuole, and the cell wall (Prasad et al., 1995; Takahama and Egashira, 1991; Van Huystee and Zheng, 1995). In the cell wall PODs are present in soluble, ionically-bound and covalently-bound forms and, in addition to a detoxicant role as scavengers of H2O2, they are involved in a number of physiological processes which regulate cell growth by catalysing the formation of cross-links between extensin and feruloyated polysaccharides and the polymerization of lignin precursors (Abeles and Biles, 1991; Christensen et al., 1998; Goldberg et al., 1983; Imberty et al., 1985; Lagrimini, 1991; Lagrimini et al., 1993; Polle et al., 1994; Sato et al., 1993). Under stress conditions, the enhanced peroxidase activity in the intercellular spaces, stimulating cell wall stiffening (Gaspar et al., 1985), probably reduces cell growth which might represent a mechanical adaptation to adverse conditions (Castillo, 1986; Ranieri et al., 1995). This kind of action has been attributed mainly to peroxidases whose activity can be detected by using, in the enzymatic assay mixture, syringaldazine as a specific substrate. There is histochemical and biochemical evidence that only cell walls that are undergoing lignification are able to oxidize syringaldazine (Christensen et al., 1998; Imberty et al., 1985).
The objective of this study was to investigate whether, in iron-deficient sunflower plants, equilibrium between the tendency to generate toxic oxygen species and the capacity to counteract them was altered. For this purpose the H2O2 content, as a bioindicator of oxidative status, and the response of iron-containing peroxidases which play a fundamental role in reducing H2O2 were analysed. Besides this, to verify whether iron deficiency induced either a generalized alteration or a differential modification in the peroxidase response, both ascorbate-dependent and non-specific peroxidase activity were analysed at an apoplastic and an intracellular level. Non-specific peroxidase activity was tested by using as the electron donor both guaiacol and syringaldazine. Biochemical and electron microscopy analyses were compared.
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Plant culture
Sterilized sunflower seeds (Helianthus annuus L. cv. Hor) were germinated in Petri dishes for 4 d and the seedlings were grown in perlite for a week. After this period, plants were transferred to hydroponic culture and separated in two groups. Control plants received half-strength Hoagland's nutrient solution containing 74 µM Fe-EDTA, and -Fe plants received the same nutrient solution except that the Fe concentration was 37 µM the first week, 18.5 µM the second and the third weeks and then iron was omitted in the fourth week. To simulate natural environmental conditions and accelerate the Fe deficiency process, 0.3 mM CaCO3 was added to -Fe plants (Iturbe-Ormaetxe et al., 1995
; Terry, 1980). Nutrient solution was renewed every 3 d. Plants were grown in 10 l plastic pots (10 plants per container) for 4 weeks in a greenhouse at 25 °C with a 14 h photoperiod and a photosynthetic photon flux density of 400 µE m-2 s-1. All chemical and biochemical analyses were carried out on fully expanded middle-aged leaves from control and treated plants.
Iron content analysis
Iron was quantified by atomic absorption spectrophotometry using a Perkin Elmer 373 spectrophotometer. Leaves were extensively washed with 0.1% Teepol detergent in 0.1 M HCl and, after being frozen in liquid nitrogen, they were lyophilized and stored under vacuum. Aliquots of 100 mg of lyophilized leaves were dissolved in 5 ml of hot H2SO4, with the addition of H2O2 until complete clarification of the sample, and the volume was adjusted to 25 ml with twice-distilled water (Jones and Wallace, 1992). Iron content was quantified using standard solutions.
For the determination of iron in the low-molecular-mass fraction (<3 kDa), leaves were homogenized with Chelex-treated 25 mM K-phosphate buffer (pH 7.0, 1:10, w:v) and centrifuged at 15 000 g for 20 min, according to the method reported previously (Iturbe-Ormaetxe et al., 1995). The supernatant was filtered through Centricon (Amicon) membranes with 3 kDa of molecular exclusion and iron was quantified using an atomic absorption spectrophotometer (AA-670G, Shimadzu) equipped with a graphite furnace atomiser (GFA-4A, Shimadzu).
Growth parameters
Leaf discs of known area were weighed to determine the leaf mass/leaf area ratio, and after being freeze-dried, weighed again to obtain the fresh weight/dry weight ratio. Soluble protein content was measured by the protein-dye binding method using bovine serum albumin as the standard (Bradford, 1976). The spectrophotometrical reading was measured at 595 nm.
Chlorophyll and carotenoid content determination
Leaf chlorophyll and carotenoid content was quantified by HPLC analysis according to the method reported earlier (Ciompi et al., 1997). Briefly, leaf discs of known area and weight were ground in a mortar with liquid nitrogen in cold 100% HPLC-grade acetone containing sodium ascorbate under dimmed green light. The mixture was filtered through 0.2 µm Minisart SRT 15 filter (Sartorious) and immediately analysed. The HPLC pigment separation was performed at room temperature with a non-endcapped Zorbax ODS column (Chrompack SA 5 µm particle size, 250x4.6 mm).
H2O2 content evaluation
H2O2 content was measured following the method reported previously (Brennan and Frenkel, 1977) based on the formation of a complex titanium-peroxide. The leaf samples, homogenized in cold 100% acetone (1:2, w:v), were centrifuged for 10 min at 10 000 g and 20% TiCl4 in concentrated HCl was added to supernatant aliquots to give a final titanium concentration of 4%, followed by the addition of NH4OH (0.2 ml for each ml of sample) to precipitate the titanium-peroxide complex. After 5 min of centrifugation at 10 000 g the resulting pellet was washed five times in acetone and then resuspended in 2 N H2SO4. The absorbance of the solution was read at 415 nm against a blank containing H2O instead of leaf extract. H2O2 content was calculated using a standard curve of H2O2 of known concentrations from 0.1–1 mM.
Preparation of the apoplastic fluid
Freshly harvested intact leaves (10 g) were rinsed with distilled water and vacuum infiltrated (-65 kPa, three cycles of 30 s each) in 50 ml of 66 mM K-phosphate buffer (pH 7) and 100 mM KCl. After having been wiped, the leaves were centrifuged at 1500 g for 10 min at 4 °C to obtain the infiltrated washing fluid (IWF) (Ranieri et al., 1996). For the enzymatic assay, this fluid was dialysed and utilized immediately for the biochemical analyses while the residual cell material (RCM) was immersed in liquid nitrogen and stored at -80 °C until use.
Separation of soluble, ionically and covalently-bound peroxidases
The separation of soluble, ionically and covalently-bound POD was performed as reported earlier (Ranieri et al., 1995). Freshly harvested leaves were homogenized at 4 °C with 66 mM Na-phosphate buffer, pH 6.1, and centrifuged at 800 g for 5 min. The supernatant was again centrifuged at 10 000 g for 5 min and the second supernatant was considered the soluble fraction. The first pellet was washed twice with phosphate buffer, twice with water and, after continuous shaking for 1 h at 4 °C 2% Triton X-100, it was again rinsed five times with water. The pellet obtained was then treated with 1 M CaCl2 for 1 h and centrifuged at 800 g for 10 min at 4 °C. The resulted supernatant was the ionically-bound fraction and the pellet was washed several times with distilled water and incubated for 16 h at room temperature with 0.3% cellulase, 0.3% macerase and 0.3% cellulolysin in 50 mM Na-acetate buffer (pH 5.5) to obtain the covalently-bound fraction, after centrifugation at 800 g for 10 min. The residual cell wall material was dried at 80 °C and weighed.
Enzyme extraction and determination
To test POD activity, RCM was homogenized with quartz-sand, liquid nitrogen and 10% (w/w) polyvinylpyrrolidone (PVP) in 220 mM TRIS-HCl (pH 7.4), 250 mM sucrose, 50 mM KCl, 1 mM MgCl2, 1% ß-mercaptoethanol, and 0.01% (w/v) phenylmethylsulphonyl fluoride (PMSF) (1:2.5, w:v) and centrifuged at 12 000 g for 30 min at 4 °C (Ranieri et al., 1997).
To test the activity of the cytoplasmic and chloroplastic enzyme markers, glucose-6-P dehydrogenase (G6PDH, EC 1.1.1.49) and glyceraldehyde-3-P dehydrogenase (GAPDH, EC 1.2.1.12), the RCM was homogenized with liquid nitrogen in a cold buffer solution (1:4, w/v) consisting of 50 mM HEPES (pH 7.4) containing 0.25 M sucrose, 70 mM KCl, 10 mM MgCl2, 1 mM EDTA, 1% (w/v) BSA, 10 mM ß-mercaptoethanol (ßME), and phenylmethylsulphonyl fluoride (PMSF) (50 µg ml-1). NH4SO2 (70% of saturation) at 4 °C was added to the supernatant obtained after centrifugation of the homogenate at 39 000 g for 15 min at 4 °C, to precipitate the proteins. After centrifugation (17 000 g for 15 min at 4 °C), the pellet obtained was resuspended in 10 mM HEPES buffer at pH 7.4, containing 10 mM MgCl2, 50 mM KCl, 0.5 mM EDTA, and 1 mM ßME (Ranieri et al., 2000) and dialysed overnight. The extract obtained was utilized for the analysis of the enzymatic activity.
For APX determination, the RCM was homogenized in a cold medium consisting of 0.1 M Tricine-KOH buffer (pH 8.0), containing 1 mM DTT, 10 mM MgCl2, 50 mM KCl, 1 mM EDTA, 0.1% Triton X-100, and PMSF (50 µg ml-1). To this medium was added 50 mM AA to maintain the enzyme active during the extraction procedure. After centrifugation at 12 000 g for 30 min at 4 °C, the supernatant was dialyzed for 2 h against 50 mM AA solution and tested for enzyme activity.
Enzyme activity assay
The incubation medium for the G6PDH activity determination consisted of 86.3 mM triethanolamine-HCl buffer (pH 7.6), 6.7 mM MgCl2, 12 mM glucose-6-P, 0.37 mM NADP+, and a suitable aliquot of IWF or RCM. The absorbance was read at 340 nm and the activity was expressed as µmol of NADP+ reduced min-1. The activity assay for GAPDH was carried out at 25 °C, recording the decreasing of the absorbance at 340 nm. The reaction mixture contained 50 mM TRIS-HCl buffer (pH 7.8), 10 mM MgCl2, 5 mM EDTA, 3 mM ATP, 100 µM NADPH, 5 mM 3-P-glycerate, 0.45 U ml-1 P-glycerate kinase, and an aliquot of RCM or IWF (Ranieri et al., 2000).
APX activity was determined following the decrease in absorbance at 290 nm due to the oxidation of ascorbic acid in the first 30 s from the start of the reaction, using the extinction coefficient of 2.8 mM-1 cm-1 for ascorbate. The reaction medium contained 0.5 mM ASA, 0.1 mM H2O2, 1 mM EDTA, and 0.1 M HEPES–KOH buffer (pH 7.8) (Ranieri et al., 1996). One enzymatic unit is equivalent to 1 µmol of ascorbic acid oxidized min-1 cm-1. To discriminate between APX and POD activities, 50 mM p-chloromercuribenzoate (pCMB), known to inactivate APX, was added to the enzymatic reaction mixture (Miyake and Asada, 1992).
POD activity was tested using two different reducing phenolic substrates, guaiacol and syringaldazine. The reaction medium for guaiacol-POD contained 20 mM Na-acetate (pH 5.0), 30 mM H2O2, 2 mM guaiacol and an appropriate amount of enzyme extract. The rate of guaiacol oxidation was recorded at 470 nm (Ranieri et al., 1997) and the activity was calculated using the extinction coefficient of 26.6 mM-1 cm-1 for guaiacol. The activity of syringaldazine-POD was determined by measuring the increase in absorbance at 530 nm of the reaction mixture containing 200 mM Na-K phosphate buffer (pH 6.0), 2 mM syringaldazine, 2.5 mM H2O2, and the protein extract (Pandolfini et al., 1992).
POD isoenzyme determination
Protein samples solubilized in 1% glycine were resolved by isoelectrofocusing (IEF) in a horizontal slab apparatus (Bio-Rad), on 5% acrylamide gel containing 5% ampholine (pH 3.5–10). The IEF run was carried out at constant voltage of 100 V for 15 min, followed by 15 min at 200 V and 30–45 min at 450 V. The POD isoenzymes were visualized by incubating gel in 0.5% benzidine and 0.03% H2O2 in 4.5% acetic acid (modified from Ros Barceló et al., 1987) and after the end of the reaction by 7% acetic acid for 30 s, gels were immediately photographed.
In situ localization of H2O2 and peroxidase activity
Freshly harvested leaves were cut in slices (1–2 mm2) avoiding the central vein and subjected to two distinct protocols for cytochemical localization of H2O2 and determination of POD activity.
H2O2 production was assessed cytochemically via determination of cerium perhydroxide formation after reaction of CeCl3 with endogenous H2O2 (Bestwick et al., 1997). Leaf slices were incubated for 1 h in 5 mM CeCl3 in 50 mM 3-(N-morpholino)propanesulphonic acid (MOPS) pH 7.2, fixed in 1.25% glutaraldehyde, 1.25% paraformaldehyde in 50 mM Na-cacodylate buffer (CAB) pH 7.2 for 1 h and washed twice in CAB buffer for 10 min (Bestwick et al., 1997).
Cytochemical analysis of POD was carried out according to Sottomayor et al. (Sottomayor et al., 1996) with some modifications as reported below. Leaf strips were fixed in 3% glutaraldehyde in 50 mM Na-phosphate buffer, pH 7.0 for 2 h at 4 °C and washed several times in 50 mM Na-phosphate buffer, pH 7.0 for 10 min. After a pre-incubation in 50 mM TRIS-HCl pH 7.6 for 10 min, tissue sections were incubated in 0.1% diaminobenzidine, 0.01% H2O2 in 50 mM TRIS-HCl, pH 7.6 for 1 h in the dark at room temperature and then washed in 50 mM TRIS-HCl, pH 7.6 for 10 min. A series of fragments incubated without H2O2 were used as blank controls.
From this point on, samples for H2O2 and POD cytochemical localization were subjected to standard procedure for electron microscopy. All samples were post-fixed for 2 h in 1% osmium tetroxide in 50 mM Na-cacodylate buffer, pH 7.2. Samples were washed twice in the same buffer, dehydrated in a graded ethanol series (25, 50, 75, 90, 100%), transferred in propylene oxide and gradually embedded in Epon-Araldite. Thin sections of embedded tissues were obtained on a Reichert-Ultracut microtome, stained with lead citrate, mounted on uncoated copper grids and observed using a transmission electron microscope (Hitachi 300, Tokyo, Japan) at 75 kV.
Statistical analysis
The whole experiment was performed twice. Values shown in the table and figures are the means of five determinations for each analysis. Comparison between means was evaluated by t-test and the P=0.05 level of error.
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Results |
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Determination of iron, chlorophyll and carotenoid content, growth parameters and H2O2 level
Four weeks after iron-deficiency was imposed, the fully expanded middle-aged leaves of sunflower exhibited symptoms of chlorosis which agreed well with the values of chlorophyll content (Table 1
). In fact, iron-deficiency induced a decrease in total chlorophyll down to approximately 16.4% of the control value, reaching a content of 7 nmol cm-2 of leaf area. Similar behaviour, but to a lesser extent, was shown by total carotenoid content (-64%) in iron-deprived leaves with respect to the control (Table 1).
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Atomic absorption spectrophotometry was used to quantify the total leaf iron content which was significantly reduced by about 22%, as compared with the control value (Table 1
). In Fe-deficient leaves the content of catalytic iron, measured in the <3 kDa fraction containing iron in the free form or bound to small molecules, was drastically decreased (-86%) in comparison to the control (Table 1).
The leaf mass/leaf area ratio decreased significantly from 19.4 mg cm-1 in the control to 16.5 mg cm-1 in iron-deficient plants (Table 1). Soluble protein content, expressed either on a leaf mass and leaf area basis (Table 1), underwent a significant decrease of 23% and 32%, respectively, following iron-deficiency, while the fresh weight/dry weight ratio did not change (Table 1).
The spectrophotometric monitoring of the titanium-peroxide complexes, which allowed quantification of total H2O2 content, indicated a significant increase (+28%) of the peroxide following iron deficiency (Table 1).
Apoplastic and intracellular peroxidase activity
The intercellular washing fluid was used to test the activity of ascorbate-dependent peroxidase (APX, Fig. 1A) and soluble non-specific-POD (Fig. 2A, D). Accidental contamination of IWF with intracellular proteins during the infiltration procedure was tested by analysing the activity of the cytosolic and chloroplastic enzyme markers G6PDH and GAPDH, respectively. Regardless of the treatment, the relative activity of these marker enzymes in the IWF was always less than 0.1% of the total activity in the RCM (data not shown). With regard to non-specific POD, neither guaiacol (Fig. 2A) nor syringaldazine-dependent peroxidase activity (Fig. 2D) changed following iron deficiency imposition, while a significant decrease was observed when the intercellular washing fluid was tested for APX specific activity (-16%, Fig. 1A).
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Abs530 min-1 mg-1 proteins, except for the CB fraction, whose activity is reported as When the ionically and covalently-bound wall fractions were tested for POD activity, a differential effect, related to the two different electron donors used, was determined by iron deficiency in sunflower plants. The activity of guaiacol-POD decreased in both the fractions analysed although to a different extent: 10% and 42% in the ionically (Fig. 2B
) and covalently-bound (Fig. 2C) wall fractions, respectively; by contrast, the peroxidase activity measured as syringaldazine oxidation decreased only when the covalently-bound wall fraction was assayed (-22%, Fig. 2F).
In contrast to what happened because of soluble guaiacol-POD in the apoplastic medium of iron-deficient leaves, at an intracellular level (RCM) these enzymes underwent a decrease of 51% compared with the control (Fig. 1C). On the other hand no difference was shown between apoplastic and symplastic APX behaviour, in fact the ascorbate-dependent activity at intracellular level also decreased (-27%) in the plants grown in iron-deprived solution (Fig. 1B).
In situ apoplastic-localization of H2O2 and peroxidase activity from electron microscope cytochemical analysis
The histochemical assays based on the reaction of H2O2 with CeCl3 produced electron-dense insoluble precipitates of cerium perhydroxides at sites where H2O2 was accumulated and produced. CeCl3 staining was present on the tangential and radial walls of epidermal cells in plants grown under iron-deficiency (Fig. 3). Particularly striking was the staining at the sites of connection between mesophyll adjacent cell walls close to the intercellular spaces.
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The 3,3'-diaminobenzidine/H2O2 technique revealed electron-dense deposits, indicative of peroxidase activity, in all samples of control leaves analysed. On the other hand, in the iron-deficient plants, peroxidase activity was almost absent in both epidermal and mesophyll cells (Fig. 4
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Electrophoretic patterns of POD isoforms
The isoenzyme profile of intracellular and cell wall fractions of POD (soluble, ionically and covalently-bound) separated on IEF gel and revealed by the benzidine staining procedure, is shown in Fig. 5. Qualitative and quantitative differences between the control and iron-deficient samples were present both at the intracellular and the cell wall level. In the iron-deprived plants, at an intracellular level, the staining of one anodic band was very much less intense in comparison to the control (Fig. 5A). Regarding the peroxidase isoform patterns of apoplastic fractions, no particular variation was evident for soluble peroxidase isoforms in the iron-deprived leaves (Fig. 5B), while the separation of the isoenzyme of the ionically-bound fraction showed the lack of one cathodic band (Fig. 5C). A more generalized decrease in the band staining intensity was especially evident for the covalently-bound fraction, where the disappearance of two cathodic bands as a result of iron deficiency could be seen (Fig. 5D).
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Discussion |
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Sunflower plants grown under iron-deficient conditions contained less chlorophyll in comparison to plants well supplied with iron (Table 1
). Such a large decrease in the chorophyll content may be ascribed to the well-known Fe requirement for the formation of precursors of the chlorophyll molecules, 
-aminolevulinic acid and protochlorophyllide (Marschner, 1986). A similar trend in the lack of carotenoids was observed, even if to a lesser extent with respect to chlorophyll content (Table1). The strong fall in chlorophyll content in the chlorotic sunflower leaves was accompanied by a decrease in the amount of iron, which was only 22% lower than in the control plants (Table 1), according to the data reported previously (Mengel, 1995). The lack of variation in the leaf fresh weight/dry weight ratio between chlorotic and control leaves seems to point to the absence of a water stress secondary to iron deficiency, so that the detected decrease in the leaf fresh weight/area ratio can probably be correlated with a minor thickness of iron-deficient leaves.
The synthesis of proteins is known to be impaired by iron deficiency and the chromoproteins of the chloroplast are particularly susceptible to degradation under such conditions (Marschner, 1986). In sunflower, the amount of leaf soluble proteins was significantly affected by iron deficiency (-23% and -32%, respectively, on a leaf mass and a leaf area basis), the extent of their decline being similar to that recorded for iron content. A similar result of a small but significant decrease in protein content was detected also by Iturbe-Ormaetxe et al. in iron-deficient pea plants (Iturbe-Ormaetxe et al., 1995).
The H2O2 content underwent a significant increase following the iron starvation treatment. Although H2O2 is not particularly detrimental to cell metabolism, it can be reduced to extremely reactive OH. radicals through the Fenton reaction in the presence of iron in the free form or bound to small molecules, such as amino acids, nucleotides and organic acids (Halliwell and Gutteridge, 1989). In iron-deficient sunflower plants, the content of catalytic iron was extremely low and, consequently, the Fenton reaction was probably unlike to occur. In fact, previous data indicating the absence of oxidative damages to lipids and proteins in iron-deficient sunflower (Ranieri et al., 1999b) suggest that sunflower plants were still sufficiently protected against oxidative stress.
The high levels of H2O2 suggest that iron starvation may induce both a decreased capacity to peroxide detoxification and/or an active production of peroxide with a consequent rise of oxidative cell status. In sunflower plants both mechanisms probably occurred. In fact, previous results (Ranieri et al., 1999b) indicated an increase in SOD activity following iron deficiency, so leading to an enhanced production of H2O2. On the other hand, the decreased capacity to detoxify overproduced H2O2 may be the result of an unsuccessful activation and/or a reduced production of ubiquitous haem-containing peroxidase enzymes. In fact, as visualized in vivo through hystochemical analysis (Fig. 4), iron-deprived sunflowers showed a decreased number of electron-dense deposits at the cell wall level with respect to the control, indicating less intense peroxidase activity at this level as a consequence of iron deficiency. The results of histochemical analysis were consistent with those from biochemical measurements. Indeed as a consequence of iron deprivation, specific APX activity was reduced in the apoplastic fluid (-16%, Fig. 1A), as well as at an intracellular level (Fig. 1B). The diminished APX activity due to iron deprivation may result as a consequence of the high request for iron from the APX molecule, as it contains, in addition to the haem group, also a non-haem iron atom. Similarly, Iturbe-Ormaetxe et al. found a marked fall in this enzymatic activity when the total extract of iron-deficient pea leaves was assayed (Iturbe-Ormaetxe et al., 1995).
In contrast to what happened for APX, neither soluble nor ionically cell wall bound syringaldazine-POD underwent any change in iron-deprived plants in comparison to control ones (Fig. 2D, E). These results seem to suggest that under iron-starvation the sunflower plants try to retain, preferentially, the functionality of the PODs mainly involved in the maintenance of cell wall structure and, in turn, an unchanged cellular homeostasis or turgor (Otter and Polle, 1994; Takahama, 1993). By contrast, while the soluble isoforms of guaiacol-POD did not show variations in their activation in either stressed or unstressed plants, the ionically and covalently cell wall-bound peroxidase activity underwent a reduction in iron-deprived plants (Fig. 2A, B, C). These data, together with the large electron-dense deposits of cerium perhydroxides detected at the cell wall level (Fig. 3), which indicate localized accumulation of H2O2, suggest a differential inactivation of POD isoforms involved in H2O2 detoxification, even though increased NADPH oxidase activity could not be excluded (Bestwick et al., 1997). However, differential susceptibility of peroxidase isoforms due to iron deficiency is very evident in sunflower plants. It is worth pointing out that an enhanced proton extrusion from strategy I plants as a consequence of iron deprivation can induce apoplast alkalinization which, in turn, would be responsible, at least partially, for diminished cell wall peroxidase activity. The apoplast pH may be further enhanced by the presence of 
anions in the nutrient solution, which, contributing to raise apoplast pH, would induce a further iron immobilization (Kosegarten et al., 1999). In addition, the apoplastic pH plays a fundamental role in the regulation of the ionic transport mechanism at the plasmalemma level. In particular, free Ca2+ would be involved in the regulation of basic POD at the cell wall level, through the removal of inhibition by low molecular weight molecules (Heath, 1988).
The separation of apoplastic POD isoforms by IEF, followed by benzidine staining (Fig. 5), has confirmed the data obtained through spectrophotometric analysis on the generalized reduction in POD activity. In fact, in addition to an overall reduction in staining intensity of the iron-deprived samples in comparison to the control ones, qualitative variations in isoenzyme patterns, mainly due to cathodic isoforms of the ionically and covalently-cell wall-bound fraction, were present.
The specific role, within the lignification process, of the different acidic and basic POD isoforms, is still uncertain. For some authors the lignification process could be associated with a stimulation of acidic isoforms (Christensen et al., 1998; Lagrimini, 1991; Lagrimini et al., 1993), while other authors have found a relationship between lignification and alkaline peroxidase activity (Abeles and Biles, 1991; Polle et al., 1994; Sato et al., 1993). In sunflower plants, the decrease of guaiacol-POD and the invariance of syringaldazine-POD activity in the IB fraction, accompanied by the lack of a cathodic isoform in the IEF pattern, may suggest that the lignification process was associated with acidic isoenzymes. However, this hypothesis on a correlation between acidic isoforms and lignification activity needs further investigations.
At the intracellular level, POD activity, which was tested by using guaiacol as the electron donor, diminished in the iron-deficient plants (Fig. 1C), as confirmed by a generalized staining reduction of bands separated by IEF (Fig. 5A), particularly evident due to an anodic form. In addition to those present in the cytosol and at the endoplasmic reticulum and the Golgi apparatus level, the unspecific PODs at the intracellular level are localized mainly in the vacuole, where only basic isoforms were found (Schloss et al., 1987). The minor compromising of the basic isoforms following iron starvation seems to indicate a preservation of H2O2 reduction activity at a vacuole level where other forms such as APX are not present.
In conclusion, the growth of sunflower plants under iron deficiency conditions affects peroxidase isoforms differently, inducing a preferential reduction in activity of those isoforms involved in the detoxification processes. As a consequence, a secondary oxidative stress, as indicated by the accumulation of H2O2, may arise. However, previous data on the absence of oxidative damage to lipids and proteins (Ranieri et al., 1999b) indicated that sunflower plants were still sufficiently protected against oxidative stress, probably due to the almost completely lack of catalytic iron capable of triggering the Fenton reaction.
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Acknowledgments |
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This research was supported by MURST-cofin 98. We are indebted to Dr Lupetti (ARPAT, Pisa) for the measurement of iron in the low-molecular-mass fraction.
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Notes |
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3 To whom correspondence should be addressed. Fax: +39 50 598614. E-mail: aranieri{at}agr.unipi.it
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