Plant Science 167 (2004) 925935

Biochemical and ultrastructural changes in leaves of potato plants grown under supplementary UV-B radiation
Isabel Santos , Fernanda Fidalgo, Jos M. Almeida, Roberto Salema
Departamento de Bot nica and Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, a Rua do Campo Alegre 823, 4150-180 Porto, Portugal Received 29 January 2004; received in revised form 16 April 2004; accepted 25 May 2004 Available online 19 June 2004

Abstract As tolerance to UV-B radiation involves many mechanisms, changes in several parameters associated to plant protection against UV-B radiation were studied in leaves of potato plants, exposed to this radiation. UV-B exposure increased constitutive avonoids and two new types were induced. Chlorophyll amount, as well as, total protein content, was slightly decreased. However, the synthesis of a 34 kDa polypeptide was induced. Leaf area diminished, leaf dry weight and leaf thickness increased, but the gross anatomy was not changed, neither was the structural integrity of the cells. All sub-cellular structures maintained their integrity although some changes were detected. In guard cells, the fractional volume of both plastids and starch was reduced, whereas thylakoids increased. The appearance of paracrystalline inclusions in peroxisomes in both epidermal and palisade cells was conspicuous. The fractional volume of both starch and chloroplasts in palisade cells decreased. The activity of the antioxidant enzymes catalase, ascorbate peroxidase and guaiacol peroxidase increased associated with the induction of a new catalase isoform and three new guaiacol isoperoxidases. Our results show that potato plants activate several defence systems, altogether contributing to the preservation of cell structural integrity, suggesting that this encompassing defence response enables plants to cope with UV-B aggression. 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Catalase; Flavonoids; Peroxidase; Potato; Ultrastructure; UV-B radiation

1. Introduction The stratospheric ozone layer is the principal agent absorbing ultraviolet radiation in the Earths atmosphere and the thinning of this layer has led to an increase in solar UV-B radiation (280320 nm) reaching the Earths surface. The impact of UV-B radiation on growth, development, biomass accumulation, yield and metabolism of plants has been studied by several research groups (references in [1,2]). Studies have revealed considerable variation in sensitivity to UV-B within, as well as between species [35]. Measurements of some physiological parameters such as the level of UV-B absorbing compounds and chlorophyll content have proved useful indicators of UV-B tolerance or sensitivity [6]. The differential sensitivity of plants is partially explained by
Corresponding author. Tel.: +351 22 607 4900; fax: +351 22 609 9157. E-mail address: isantos@ibmc.up.pt (I. Santos).

their ability to respond to UV-B through the induction of defensive pathways plus increases in protective pigments and in leaf thickness [2,710]. There is evidence that avonoids reduce damage from UV-B radiation because they act as UV lters, reducing the penetration of potentially damaging UV-B radiation. Mutants of Arabidopsis lacking avonoid production are hypersensitive to UV-B radiation whereas an Arabidopsis mutant possessing constitutive elevated accumulation of avonoids and other phenolics is tolerant to lethal UV-B level [9,11]. Plant capability to accumulate UV-B absorbing compounds and the readiness of this accumulation has been correlated with UV-B tolerance [12]. Several recent studies have shown that UV-B radiation cause increase in the level of cellular reactive oxygen species generating oxidative stress and it is generally accepted that the mechanism of UV-B toxicity involves oxidative damage (review in [13,14]). Superoxide dismutase, ascorbate peroxidase and catalase are important enzymes that protect plants against oxidative damage. The behaviour of this antioxidant

0168-9452/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.05.035

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system in UV-B exposed plants has begun to be evaluated [1518] and some studies showed similarities between responses to UV-B radiation and other oxidative stress such as ozone treatment [15,19]. At present, our knowledge concerning the role of the antioxidant systems in protecting plants under UV-B stress is limited because few data exist on this matter and studies have been made on very few plant species. Information on UV-B damage to mesophyll cells analysed at the ultrastructural level is also limited [8,2022] and the available data indicate considerable differences in the level of cell damage. Experiments designed to nd out whether UV-B physiological damage is necessarily paralleled by changes in the cell ultrastructure have not been performed. Due to the broad range of UV-B radiation targets plants have evolved diverse protection mechanisms that help them to cope with the UV-B exposure. Using different approaches and analysing different parameters a better knowledge of the importance of parameters associated with the UV-B tolerance could be achieved. Solanum tuberosum is the most important non-cereal food crop and Krupa and Kickert [3] suggest it to be tolerant to UV-B radiation and sensitive to ozone; unfortunately the biochemical and molecular mechanisms underlying this behaviour is unknown. As a matter of fact, physiological, biochemical, and ultrastructural changes induced by UV-B radiation have not been studied in this species indeed. The aim of the present work was to screen a variety of parameters, considered to play an important role in plant protection against UV-B radiation [2,5,6,7,23], to get an encompassing view of the way this important crop plant stands when exposed to enhanced levels of UV-B radiation. We have analysed changes in leaf area and leaf thickness, quantitative and qualitative changes in protein and avonoid composition and the change in chlorophyll levels, in potato leaves exposed to UV-B radiation. Catalase, ascorbate peroxidase and phenol-oxidizing peroxidase behaviour was also analysed along with the effects of UV-B radiation on cell ultrastructure of the adaxial side of leaves.

ized at 300 nm [24]. Plants were alternately watered with Hoaglands solution and with deionized water and they were kept under described conditions until the end of their life cycle. After 4 and 8 days of exposure to UV-B radiation, leaf discs were punched out from the apical leaets from previously labelled leaves (fully expanded leaves) and used for ultrastructural studies. After 8 days of exposure to UV-B the previously labelled leaves were collected and frozen in liquid nitrogen and stored at 80 C for biochemical studies. To minimise plant-to-plant variation three to four plants were sampled and pooled. In general, four such samples were taken during independent experiments. The biochemical results presented represent the average over three independent experiments. 2.2. Growth parameter measurements Leaf thickness was measured with a photomicroscope tted with a micrometer employing 25 semi-thin transverse sections cut from leaf material prepared for electron microscopy studies pertaining to three different plants. In addition, 20 thin fresh leaf sections obtained from four different leaves were also used. Leaf water content was determined using some of the leaf disks, which were dried in an oven at 70 C until to a constant weight was obtained; then, the dry matter was derived. The height of the plants was measured at ower initiation. Plant height was measured from the rst node to the emerging ower stalk (n = 24). At ower initiation the leaves were collected for the measurement of projected area (n = 16). 2.3. Electron microscopy and morphometric evaluation After 4 and 8 days of UV-B exposure leaf samples were xed in glutaraldeheyde (2.5%) followed by osmium tetroxide (2%) using NaOH-PIPES buffer [25] and routinely embedded in Epon. The stereological study was done as previously described [26]. Leaf samples were collected from three different plants and ve blocks made from each. To assure random pictures for morphometric studies, three blocks were chosen and from each several ultra thin sections were cut and mounted in grids with 100 square mesh. Photographs were taken from both mesophyll and guard cells, giving a total of 36 proles of mesophyll chloroplasts and a total of 12 guard cell chloroplast proles. Morphometric determinations of fractional volume (Vv) were performed by recording the number of hits of a 1 cm2 grid on the structure under study, on pictures taken at random and enlarged to a nal magnication of 20,000. The results are average over two independent experiments and are expressed as percentage S.E.M. (n = 72 mesophyll chloroplast proles and n = 24 guard cell chloroplast proles). The polyclonal primary antibody used for immunolocalization of catalase (CAT) was raised in rabbit (Eurogentec, Belgium) against two peptides (FPSRFDPCRPAEQYP; KQAGERYRSWESDR) selected as antigenic regions from the aminoacid sequence

2. Materials and methods 2.1. Plant material Potato tubers (S. tuberosum L. cv. Dsire) were sprouted in a vermiculite:quartz sand mixture and grown in a growth chamber under an 11 h photoperiod (450 mol m2 s1 of PAR) at 26 C/15 C day/night temperature. A very homogeneous emergence of the plants was noted and 17 days after emergence, in each plant, the fourth leaf from the apex was labelled. Plants were divided into two sets, one batch served as controls, the other received equivalent PAR (450 mol m2 s1 ) supplemented with UV-B radiation as previously reported [17,21]. The biologically effective level of UV-B radiation was 7.83 kJ m2 day1 as weighted by Caldwells generalized plant action spectrum normal-

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deduced from a S. tuberosum L. cDNA for CAT2 isolated in our laboratory (NCBI accession no. AY500290). For immunoelectron microscopy studies, leaf pieces were xed in a mixture of 4% formaldehyde, 0.2% glutaraldehyde, 2% sucrose and 0.05% CaCl2 in 1.25% PIPES buffer, pH 7.2 for 1 h, at room temperature. Leaf pieces were then washed two times in 2.5% PIPES buffer with 1 mM glycylglycine, dehydrated and embedded in Spurr. Ultrathin sections were collected on formvar/carbon-coated nickel grids. Sections were etched with saturated sodium metaperiodate for 10 min, rinsed in double-distilled water and then in 0.1 M HCl for 10 min. Immunogold labelling was done by oating the grids with section side down on drops of the following solutions: (1) T1 buffer (10 mM Tris buffer containing NaCl 0.8%, pH 7.5), 10 min; (2) T2 buffer (10 mM Tris buffer, pH 7.5 containing 0.8% NaCl, 0.2% Tween 20 and 1% bovine serum albumin-globulin free, 30 min; (3) antibody anti-catalase diluted in T2 buffer (1:25), overnight at 4 C, in a moist chamber; (4) T2 buffer, 6 5 min; (5) 10 nm colloidal gold-goat rabbit IgG (AuroProbeTM , Amersham) at a dilution of 1:25 in T2 buffer, 90 min; (6) T2 buffer, 3 5 min; (7) double-distilled water, 3 5 min. Controls were done by omitting incubation with antibody anti-catalase or by substituting the primary antibody with a similar amount of pre-immune rabbit serum. 2.4. Pigment assays Chlorophylls were extracted and quantied as described previously [21]. UV-B screening pigments were extracted in methanol:HCl:water (79:1:20, v/v). After centrifugation (3000 g for 10 min), the absorbance of the supernatant was recorded between 250 and 500 nm. For the extraction of the avonoids leaf material was ground in 90% acetone using a pre-chilled mortar and pestle, then extracts centrifuged at 15,000 g for 15 min. Chlorophylls and carotenoids were extracted from this acetonic supernatant using petroleum ether (PE: 4060 C) followed by diethylether. The acetone solution was evaporated to dryness under vacuum and the residue dissolved in a minimum volume of methanol. Aliquots were used for two-dimensional chromatography using thin layer cellulose plates (Merck 5718) with n-butanol:acetic acid:H2 O (4:1:5) as the rst solvent and 5% acetic acid as the second solvent. The plates were observed under visible and ultraviolet light. 2.5. Protein analysis Leaf disks were ground at 4 C in TrisHCl (60 mM, pH 6.8) using a pre-chilled mortar and pestle. The extract was centrifuged (15 min at 4000 g) and the supernatant used for protein quantication (soluble protein) according to Peterson [27]. The pellet was re-suspended in the same buffer containing sodium dodecyl sulphate (SDS) 2.5% (w/v) in order to extract the insoluble proteins (suspension incubated for 3 h at 4 C followed by 1 h at room temperature). The

suspension was then centrifuged for 15 min at 4000 g and proteins quantied via the same method. One-dimensional SDSPAGE of proteins was carried out as described by Laemmli [28] on 10% polyacrylamide slab gels, the wells were loaded with 10 g protein and run in parallel with the standard proteins. Gels were silver stained [21] and then analysed using a Cybertech CS1 (Berlin, Germany). Two gels were performed for independent experiments and the representative results from six gels obtained with material from the three experiments are presented. 2.6. Extraction of catalase and peroxidase Leaf material was ground at 4 C in sodium phosphate 100 mM (pH 7.3) containing 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail [one tablet of the protease inhibitor cocktail (Complete; Mini, EDTA-free; GmbH, Germany) in 10 ml extraction medium] and 2% (w/v) insoluble polyvinylpolypyrrolidone. For the analysis of ascorbate peroxidase, the extraction buffer contained 5 mM ascorbate. Homogenates were centrifuged at 19,000 g for 18 min at 4 C. To the supernatant to be used for catalase zymograms, dithiothreitol was added (10 mM nal concentration) as well as glycerol to a nal concentration of 40% (v/v). To the supernatant to be used for ascorbate peroxidase activity study glycerol was added to a nal concentration of 40% (v/v). Ascorbate peroxidase gels were run in the extraction day. Measurement of protein levels in the supernatants before addition of glycerol was performed according to Peterson [27]. 2.7. Detection of catalase and peroxidase activity Catalase (CAT; EC 1.11.1.6) isozymes were separated on non-denaturating polyacrylamide gels, as described by Laemmli [28] with SDS omitted and the gels supported by 10% glycerol. Electrophoretic separation and staining for catalase activity was performed according to Clare et al. [29]. Ascorbate peroxidase (APX; EC 1.11.1.11) isozymes were separated on non-denaturating polyacrylamide gels as described for catalase except that the carrier buffer contained 2 mM ascorbate. The gels were pre-run for 30 min to allow ascorbate present in the carrier buffer to enter the gel after which the samples were applied. Following electrophoretic separation, gels were stained for APX activity according to Mittler and Zilinskas [30]. In brief, gels were equilibrated with 50 mM potassium phosphate buffer (pH 7.0) containing 2 mM ascorbate for 30 min. The gels were incubated in buffer containing 4 mM ascorbate plus 2 mM H2 O2 for 20 min and then gently agitated in 50 mM potassium phosphate buffer (pH 7.8) containing 28 mM tetramethyl ethylene diamine and 2.45 mM nitroblue tetrazolium. After 1015 min the activity reaction was stopped by washing the gels with water. For both CAT and APX two gels were performed for independent experiments and the representative results from six gels obtained with material from the three experiments are presented. Gels of CAT and

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APX were scanned and the intensity of each isoform of the enzymes quantied using image analysis software KDS1 (Kodak Digital Science 1D, Eastman Kodak Company). Phenol-oxidizing peroxidase (guaiacol-peroxidase; POD, EC 1.11.1.7) isozymes were separated by isoelectric focusing (IEF) using at-bed electrophoresis apparatus (HE 950 Isobox, Hoefer Scientic Instruments) employing polyacrylamide gel Servalyt Precotes (Serva, Heidelberg, Germany). The procedure was performed according to Servalyt Precotes instructions. The pI markers were co-electrophoresed to estimate the pI of the isoperoxidases. For peroxidase activity staining, after electrophoresis, the gels were immersed into 250 mM potassium acetate buffer (pH 5.2) containing 1 mM o-dianisidine and the reaction initiated with the addition of H2 O2 at a nal concentration of 5 mM. After 30 min incubation in darkness at room temperature, the gels were washed with distilled water; pI values were determined using IEF marker kits (Serva 39211). Proteins were stained with Coomassie Brilliant Blue R (0.115% in 25% ethanol, 8% acetic acid). Gels were scanned and the intensity of bands analysed with image analysis software KDS1 (Kodak Digital Science 1D). 2.8. Statistical analysis Values presented in the text indicate mean values S.E.M. of three separate experiments. The signicance of differences between control and UV-B exposed material was analysed using the Students t-test for comparison of means at the level of signicance of P < 0.05. 3. Results 3.1. Growth parameters In comparison with the control plants, UV-B exposure signicantly increased leaf thickness; a mean of 192.5 1.55 m for the control leaves and a mean of 232.5 1.30 m for UV-B exposed leaves was quantied, difference that can be observed in Fig. 1A and B. After 8 days of UV-B

exposure the leaf dry weight was increased due to decrease in water content thus a mean of 125 1.08 mg d.w. g1 f.w. in controls and a mean of 154 1.67 mg d.w. g1 f.w. in UV-B exposed leaves was determined. UV-B exposure reduced signicantly leaf area, which was smaller by ca. 24%. The height of the plants measured at owering initiation showed that, at this time, UV-B treated plants were shorter than controls by about 28%. 3.2. Structural changes In comparison with controls, leaf gross anatomy was not changed by UV-B exposure, as observed by light microscopy of cross sections (Fig. 1A and B). Since the adaxial surface of the leaves received a higher level of UV-B radiation than the abaxial surface, ultrastructural study was focused on cells of adaxial side. In comparison with the control cells, no remarkable deviation was observed. Actually, UV-B radiation did not cause any change of the basic structural integrity of the cells nor disruption of either cell organelles or cell structures. Fig. 2A and B are representative of the ultrastructure of guard cells from control and UV-B exposed leaves. Each guard cell had a vacuolar system formed by multiple vacuoles with intact tonoplast; within the cytoplasm, the cell components appeared with typical organization and arrangement. Epidermal and mesophyll cells had a large central vacuole surrounded by a peripheral layer of cytoplasm limited by plasmalemma adpressed against the cell wall. The cytoplasm of these cells had the organelles and structures common to this type of cells. Detailed ultrastructure observations showed that UV-B exposure caused some changes in guard cells, epidermal cells and palisade cells. In guard cells, a deviation from the control cells was observed in plastids, which contained more thylakoid membranes and smaller starch granules (Fig. 2A and B). Electron microscopy observation of numerous plastid proles indicated a reduction in plastid size, decrease that was analysed through morphometric determinations. Morphometric analysis of numerous electron microscope images of plastid proles revealed a reduction of about 21% in fractional volume of the guard cell plastids (Table 1). The fractional

Fig. 1. Transverse section of potato leaves. In comparison with control (A), UV-B exposed leaf (B) appeared thicker but the gross anatomy was maintained (scale bar for A and B: 1 m).

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Fig. 2. Region of adaxial guard cells of control (A) and UV-B exposed leaves (B). Note the vacuolar system formed by multiple vacuoles with intact tonoplast (arrow). In UV-B exposed guard cells, plastids (arrowhead) appeared with more thylakoids and smaller starch granules (scale bar for A and B: 1 m). High magnication of mitochondria showing swelled cristae (compare with the inset in A) (scale bar for insets A and B: 0.5 m).

volume of starch relative to guard cell plastid size decreased (Table 1), indicating a decrease in starch content in UV-B exposed leaf. In addition, guard cell mitochondria cristae appeared swollen (Fig. 2A and B). In epidermal cells, the most conspicuous difference was observed in the peroxisomes that showed a large paracrystalline inclusion (Fig. 3) not found in control leaves. Plastids of epidermal cells of UV-B exposed leaves displayed variable degree of stromal vesiculation with no discernible pattern from cell to cell. In the palisade cells of UV-B exposed leaves peroxisomes also contained large paracrystalline inclusions (Fig. 4A and B), contrasting with controls in which such inclusions were not detected (Fig. 5A and B) even after scanning many ultrathin sections. After incubating ultrathin sections of UV-B exposed leaves with an antibody anti-CAT2, the paracrystalline inclusion in peroxisomes appeared densely labelled with
Table 1 Inuence of UV-B radiation on the fractional volume (Vv) of the chloroplasts and of the starch in guard and mesophyll cells Vv (chloroplast, grid) Control Guard cells Mesophyll cells UV-B Guard cells Mesophyll cells 52.36 4.27 47.76 6.10 41.15 2.18 32.90 5.09 Vv (starch, chloroplast) 8.32 1.03 5.71 1.10 3.87 1.10 3.47 0.72

gold particles (Fig. 6A). The immunogold labelling in peroxisomes was restricted to paracrystalline inclusion. Very few gold particles appeared occasionally scattered over other cell structures and therefore considered as background. Control sections run with pre-immune rabbit serum had no signicant gold labelling on any component of the cells (Fig. 6B) indicating the specicity of the labelling for catalase. The immunogold labelling results with the anti-CAT2 indicated that this isoform is a component in the paracrystalline inclusions in peroxisomes. UV-B exposure did not affect thylakoid system but reduced the size of starch grains in the chloroplasts of palisade cells as did in epidermal cells. Morphometric study showed a decline in fractional volume of starch already detectable after 4 days of UV-B exposure (Vv = 6.02 1.07% in controls versus 4.62 0.67% in UV-B exposed leaves) although the difference became greater by the eighth day of UV-B exposure (Table 1). At this time, the fractional volume of starch relative to chloroplast size was about 40% lower than that observed in controls. As observed in guard cells, the fractional volume of palisade cell chloroplasts was signicantly reduced (ca. 30%) after 8 days of UV-B exposure (Table 1). 3.3. Leaf pigments and proteins Since the water content in leaves was affected by UV-B exposure the level of the total chlorophylls and total proteins was expressed on both dry weight and fresh weight basis. There was no difference on total chlorophyll content between controls and UV-B exposed leaves (control = 3.05

After 8 days of exposure the fractional volume was decreased in both guard and mesophyll cells. Mean values are expressed as percentage S.E.M., and they are signicantly different from the control; P < 0.05.

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Fig. 3. Leaves exposed to UV-B radiation for 4 days. Region of epidermal cells showing well preserved sub-cellular structures; the presence of a paracrystalline inclusion in peroxisomes (arrowhead) is induced by UV-B exposure (scale bar: 1 m). Inset: detail of mitochondria after 8 days of UV-B exposure displaying alteration of their cristae, which appeared highly swollen (scale bar: 0.5 m).

Fig. 4. (A) Region of palisade cell of UV-B exposed leaves showing well preserved cytoplasm and organelles such as chloroplasts containing starch (St), mitochondria (arrow) and peroxisomes (arrowhead). (B) Detail of palisade cell showing two peroxisomes with paracrystalline inclusion (arrowhead) (scale bar for A: 0.5 m; scale bar for B: 0.25 m).

Fig. 5. (A) Region of palisade cell of control leaves showing chloroplasts containing starch (St). (B) Detail showing peroxisome with less electron dense matrix than those observed in UV-B exposed leaves and devoided of paracrystalline inclusion (compare with Fig. 4B) (scale bar for A: 0.5 m; scale bar for B: 0.25 m).

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Fig. 7. (A) 2D chromatogram of the avonoids extracted from UV-B exposed leaves showing ve spots, two of them appearing de novo (arrowhead) and other three increased in levels in contrast with the 2D chromatogram of the avonoids extracted from control leaves (B), that show only three spots with lower staining intensity than their counterparts as can be perceived comparing A with B.

Fig. 6. Immunogold localization of catalase in palisade cell of UV-B exposed leaves. (A) Abundant gold labelling over the paracrystalline inclusion in peroxisome as a result of ultrathin sections incubation in anti-CAT2 antibody. Background gold particles (encircled) on cell wall (cw) cytoplasm and plastid (p) are present in a non-signicant number. (B) Pre-immune control; a background of a few scattered gold particles (encircled) can be seen (scale bar for A: 0.25 m; scale bar for B: 0.25 m).

21.08 mg g1 d.w. in controls and 792.46 8.81 mg g1 d.w. in UV-B exposed leaves. The analysis of electrophoretic pattern of soluble proteins showed that UV-B exposure induced the appearance of a new polypeptide with a molecular mass of 34 kDa (Fig. 8).

0.21 mg g1 f.w.; UV-B = 2.87 0.19 mg g1 f.w.), considered on a fresh weight basis but a signicant decrease appeared in UV-B exposed leaves when expressed on a dry weight basis (24.43 1.76 mg g1 d.w. in controls and 18.76 1.12 mg g1 d.w. in UV-B exposed leaves). Differences were found in the absorbance patterns of methanol extracts containing the avonoid pigments as well as other UV-B absorbing compounds from control and UV-B exposed leaves. The latter showed higher absorbance when scanned between 250 and 400 nm. Flavonoid analysis by 2D chromatography showed that UV-B exposure caused accumulation of constitutive avonoids and the induction of two new avonoid types (Fig. 7, arrowheads). Total protein content was affected and a slight decrease was found when protein level was expressed on a dry weight basis; values found were 865.28

Fig. 8. SDSPAGE of soluble proteins. In comparison to control leaves (lane C), in gels of UV-B exposed leaves (lane T) a new polypeptide heavily stained, with a molecular mass of 34 kDa, was detected (arrowhead). Equal amounts of protein were loaded on the gels. Lane M: protein markers.

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activity was also increased by UV-B exposure. Analysis of staining intensity on native gels loaded with equal amounts of protein revealed that increase in catalase activity was associated with a rise in the existing isoform and the induction of a new isoform with lower mobility (Fig. 9C).

4. Discussion In potato, UV-B radiation, decreased plant height, and leaf area, and increased leaf thickness. A similar response has been reported previously for other species in response to levels of UV-B radiation similar to those employed in the present study [1,5,12,21,3134]. Due to diverse targets of UV-B radiation, plants have evolved different protection mechanisms. In potato, we found that exposure to UV-B radiation increased constitutive avonoid levels and induced appearance of two new avonoid types. Flavonoid accumulation is regarded as a key defence mechanism in higher plants against UV-B radiation. A strong correlation between avonoid accumulation and UV-B tolerance has been shown for several plant species [2,12,20,21,35,36]. Recently, an Arabidopsis mutant tolerant to lethal UV-B levels showed constitutively elevated accumulation of avonoids and other phenolics [9]. There are reports correlating the tolerance of the species to their ability to increase leaf thickness in response to UV-B exposure [5,7]. Our results show that potato plants responded to UV-B radiation developing thicker leaves and increasing in them the level of avonoids, changes that are both considered important responses of UV-B tolerant plants. However, in UV-B sensitive plant species leaf thickness and UV-B absorbing compounds may increase as well under enhanced UV-B radiation [1,2] showing that increase in leaf thickness and in UV-B absorbing compounds does not necessarily imply tolerance to UV-B radiation. The amount of total proteins was slightly decreased but the synthesis of a novel 34 kDa polypeptide occurred. Similarly, accumulation of a 34 kDa protein in potato subjected to progressive water decit, exposed to low temperatures, grown upon high salt conditions or subjected to high illumination was reported [3739]. It has been proposed that this drought-induced stress protein participates in the stabilisation of thylakoids preventing damage resulting from osmotic or oxidative stress [38]. In our material, the induction of the 34 kDa polypeptide can be considered as response to the oxidative stress induced by UV-B exposure and can be admitted that this polypeptide can contribute to the maintenance of chloroplast integrity we observed. There is little information on the effect of UV-B radiation on plant cell ultrastructure [40]. Similar ultrastructural changes have been reported for barley and pea [20,22]. In pea plants, a sequence of ultrastructural changes triggered by UV-B exposure was observed. After 1 day no signicant changes in chloroplast structure were observed whereas after 4 days, chloroplasts appeared with a disoriented lamellar system, large starch

Fig. 9. (A) Guaiacol-peroxidase isoforms from control potato leaves (lane C) and from UV-B exposed leaves (lane T) analysed by IEF gel (pH 310). In UV-B exposed leaves one anionic and two cationic isoforms appeared de novo (arrow) and all the other cationic isoforms were enhanced by UV-B exposure (lane M: pH markers, local of application). (B) APX isoform patterns from control (lane C) and UV-B exposed leaves (lane T). Five isoforms are revealed in control, isoforms 2 and 3 appearing as very faint bands. In zymogram from UV-B exposed leaves one isoform was missing and the level of the staining intensity of the isoforms 13 appeared increased compared to control. (C) CAT response to UV-B exposure. In gel of controls (lane C) only one isoform (2) was detected whereas in gels of UV-B exposed leaves (lane T) the level of that isoform was enhanced and other isoform (1) was revealed.

3.4. Peroxidase and catalase The examination of IEF patterns for guaiacol-peroxidase (POD) revealed a number of changes that occurred in response to UV-B radiation. Equal amounts of protein from both control and treated leaves generated a different number of isoperoxidases on gels, indicating de novo synthesis of new isoforms under UV-B radiation. One new isoform was anionic and focused at pH 3.05, the others were cationic focused at pH 8.1 and 8.3, respectively (Fig. 9A, arrow). Besides this change, in gels of treated leaves all the bands appeared thicker than their counterparts in controls, reecting an increase in the isoform levels induced by UV-B exposure. Ascorbate peroxidase activity was changed by UV-B exposure (Fig. 9B). In zymograms from control leaves ve isoforms were detected, three of them appearing as very faint bands (1, 2 and 3 in Fig. 9B). In zymograms from UV-B exposed leaves those three isoforms were enhanced, and of the other two isoforms one was missing and the other did not change (band 5 in Fig. 9B). Staining intensity analysis indicated that, in UV-B exposed leaves, despite disappearance of one isoform, APX activity increased, due to the rise in three of the four expressed APX isoforms. Catalase

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grains and envelope disruptions [22]. A previous study [41] in a different cultivar of pea also reported damage of the chloroplasts that progressively increased with the length of the UV-B exposure. A study in several rice cultivars showed that UV-B susceptible cultivars were severely damaged in a short period of time whereas in UV-B tolerant cultivars the damage of the cells was smaller after prolonged UV-B exposure [8]. In maize leaf exposed to UV-B, the integrity of both mesophyll and bundle sheath cells were maintained, alterations occurred in mitochondria, and in the amount of starch in bundle sheath chloroplasts [21]. In the cultivar of potato used in our studies, UV-B exposure did not result in changes in leaf gross anatomy nor in loss of the basic structural integrity of the leaf cells as well as no rupture of either chloroplast or mitochondria envelope. This response differs from the reports for other species since only minor ultrastructural changes were induced by UV-B exposure. The decrease in the fractional volume of chloroplasts can be, at least, partly attributable to osmotic adjustment; similar behaviour was found in plants exposed to water stress [26,42]. The decrease in the fractional volume of starch relative to chloroplasts reects a starch decline. Studies in pea and maize had indicated an increase in starch caused by UV-B [21,22]. The accumulation of starch in pea was interpreted as a consequence of the disruptions in the chloroplast envelope [22] whereas in maize was admitted to be a consequence of the damage of mitochondria [21]. The different response in potato, with regard to starch amount, could be due to the decrease in chlorophyll level since photosynthetic carbon assimilation is dependent on the light-harvesting capacity. In potato leaves, at ultrastructural level, the most evident consequence of UV-B exposure was the appearance of paracrystalline inclusions in peroxisomes in both epidermal and mesophyll cells. Studies done by Tenberge et al. [43] in potato and sunower peroxisomes led them to suggest that the paracrystalline inclusions in peroxisomes of these plants are made of active catalase. Further studies in sunower cotyledon peroxisomes showed that these inclusions were made up by accumulation of a catalase isoform different from the catalase in the peroxisomal matrix [44]. In our material the increase in catalase activity caused by UV-B exposure was paralleled with the appearance of crystalline inclusions in peroxisomes leading us to admit that these inclusions are very likely catalase accumulations. The immunogold labelling we observed in the paracrystalline inclusions in the peroxisomes (Fig. 6A) substantiated that hypothesis. We observed that antibody anti-CAT2 detected catalase in the inclusions of peroxisomes but not in the peroxisomal matrix indicating that this isoform of catalase (CAT2) is a protein component in these paracrystalline inclusions, similarly to reported for peroxisomes of sunower [44]. Therefore the immunogold labelling we observed using anti-CAT2 is considered good evidence of an enrichment in catalase in these cell structures in accordance to the increase in CAT2 activity induced by UV-B as seen in gels (Fig. 9C).

Several studies reported that UV-B radiation produces oxidative stress [14,16,38]. However, there is limited information on the antioxidant response of the plants to UV-B radiation and the available data indicate differences between plant species [13,16,18,38,45]. Superoxide dismutase, peroxidase and catalase are key enzymes of the antioxidant defence system. Superoxide dismutase accelerates the conversion of superoxide to hydrogen peroxide, catalase and peroxidase catalyse H2 O2 breakdown. Our previous studies showed that in the potato plants used for the present studies, the superoxide dismutase was increased by UV-B exposure [17]. The present work shows that UV-B exposure also increased the activity of CAT, and APX. The rise in these three antioxidant enzymes indicates that potato plants respond to UV-B radiation through activation of the antioxidant defence system required for control of endogenous superoxide and H2 O2 level providing cells with protection against deleterious effects of the activated oxygen species. Studies concerning the response of the antioxidant enzymes altogether in plant species exposed to UV-B radiation are scarce and the available data showed differences among the studied species. In Arabidopsis thaliana UV-B increased superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase activities and had no effect on CAT activity [15]. Activities of superoxide dismutase and ascorbate peroxidase were increased in pea leaf exposed to UV-B as well as in both green and etiolated buds [16,45]; the authors did not study catalase behaviour. In sunower, UV-B radiation decreased superoxide dismutase activity, increased the activity of CAT and did not change APX [18]. In sorghum UV-B decreased CAT activity and phenol-peroxidase activity was increased [46] whereas in maize superoxide dismutase and CAT activities were increased and APX decreased [17,40]. In Nicotiana plumbaginifolia, UV-B radiation repressed mRNA accumulation of one CAT isoform and increased transcripts of two other isoforms. Transcript levels of ascorbate peroxidase and superoxide dismutase were affected little or not at all [47]. The response of potato to UV-B radiation differs from the behaviour reported for other species since superoxide dismutase, CAT and APX activities were all increased. In sunower cotyledons, UV-B exposure increased guaiacol-peroxidase activity, response considered important for protection against UV-B stress [18]. We can consider that a similar role can be done by phenol-oxidizing peroxidase in potato since we observed an increase of this enzyme with synthesis de novo of three isoperoxidases (Fig. 9A). The increase in superoxide dismutase, APX, CAT and POD confers to potato plants an increased capacity for oxygen radical scavenging and maintenance of the integrity of cellular membranes and of all sub-cellular structures. Besides, the increase in avonoids as well as in other UV-B absorbing compounds, and in leaf thickness also provides protection against harmful effects of UV-B radiation. The UV-B-induced-polypeptide is another parameter that may also contribute to the potato plant defence. Data of the present study show that potato plants exposed to

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I. Santos et al. / Plant Science 167 (2004) 925935 solar ultraviolet-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses, Plant Cell Environ. 22 (1999) 6170. S.A.H. Mackerness, C.F. John, B.R. Jordan, B. Thomas, Early signalling components in ultraviolete-B response: distinct roles for different reactive oxygen species and nitric oxide, FEBS Lett. 489 (2001) 237242. M.V. Rao, G. Paliyath, D.P. Ormrod, Ultraviolet-B and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana, Plant Physiol. 110 (1996) 125136. S.A.H. Mackerness, B.R. Jordan, B. Thomas, Reactive oxygen species in the regulation of photosynthetic genes by ultraviolet-B radiation (UV-B: 280320 nm) in green and etiolated buds of pea (Pisum sativum L.), J. Photochem. Photobiol. B: Biol. 48 (1999) 180188. I. Santos, J. Almeida, R. Salema, The inuence of UV-B radiation on the superoxide dismutase of maize, potato, sorghum, and wheat leaves, Can. J. Bot. 77 (1999) 7076. H. Costa, S.M. Gallego, M.L. Tomaro, Effect of UV-B radiation on antioxidant defense system in sunower cotyledons, Plant Sci. 162 (2002) 939945. H. Savenstrand, M. Brosche, A. Strid, Regulation of gene expression by low levels of ultraviolet-B radiation in Pisum sativum: isolation of novel genes by suppression subtractive hybridisation, Plant Cell Physiol. 43 (2002) 402410. M. Tevini, J. Braun, G. Fieser, The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation, Photochem. Photobiol. 53 (1991) 329333. I. Santos, J. Almeida, R. Salema, Plants of Zea mays L. developed under enhanced UV-B radiation. I. Some ultrastructural and biochemical aspects, J. Plant Physiol. 141 (1993) 450456. J. He, L.-K. Huang, W.S. Chow, M.I. Whitecross, J.M. Anderson, Chloroplast ultrastructure changes in Pisum sativum associated with supplementary ultraviolet (UV-B) radiation, Plant Cell Environ. 17 (1994) 771775. J.L. Smith, D.J. Burritt, P. Bannister, Shoot dry weight, chlorophyll and UV-B-absorbing compounds as indicators of a plants sensitivity to UV-B radiation, Ann. Bot. 86 (2000) 10571063. M.M. Caldwell, Solar UV irradiation and the growth and development of higher plants, in: A.C. Giese (Ed.), Photobiology, Academic Press, New York, 1971, pp. 131177. R. Salema, I. Brando, The use of PIPES buffer in the xation of plant cells for electron microscopy, J. Submicr. Cytol. 5 (1973) 79 96. I. Santos, R. Salema, Effects of hydration conditions on the stroma inclusion of some CAM chloroplasts, Plant Cell Environ. 7 (1984) 541544. G.L. Peterson, A simplication of the protein assay method of Lowry et al. which is more generally applicable, Anal. Biochem. 83 (1977) 346356. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680685. D.A. Clare, M.N. Duong, D. Darr, F. Archibald, I. Fridovich, Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase, Anal. Biochem. 140 (1984) 532537. R. Mittler, B.A. Zilinskas, Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbato-dependent reduction of nitroblue tetrazolium, Anal. Biochem. 212 (1993) 540546. N.S. Murali, A.H. Teramura, S.K. Randall, Response differences between two soybean cultivars with contrasting UV-B radiation sensitivities, Photochem. Photobiol. 48 (1988) 653657. Y.-P. Cen, J.F. Bornman, The response of bean plants to UV-B radiation under different irradiances of background visible light, J. Exp Bot. 41 (1990) 14891495. Y-P. Cen, J.F. Bornman, The effect of exposure to enhanced UV-B radiation on the penetration of monochromatic and polychromatic

UV-B radiation undoubtedly activate several defence mechanisms, the existing and new ones, altogether contributing to the maintenance of the structural integrity of the leaf cell components. Our results suggest that the encompassing defence response is of utmost importance for plants allowing them to attain the reproductive stage and to produce tubers.

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Acknowledgements We gratefully acknowledge support from Fundao para a Ci ncia e Tecnologia (Lisboa, Portugal)project e POCTI/1999/BME/33044.

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