Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves

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Copyright ª Physiologia Plantarum 2008, ISSN 0031-9317

Physiologia Plantarum 132: 407–416. 2008

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves Sonja Veljovic-Jovanovica,*, Biljana Kukavicaa and Flavia Navari-Izzob a

Center for Multidisciplinary Studies, Belgrade University, Kneza Visˇeslava 1a, 11030 Belgrade, Serbia Dipartimento di Chimica e Biotecnologie Agrarie, Universita` degli Studi di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy


Correspondence *Corresponding author, e-mail: [email protected] Received 27 October 2007 doi: 10.1111/j.1399-3054.2007.01040.x

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o-diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent Km values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na3N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.

Introduction A small number of higher plant species, not closely related, have adapted to environments with rapidly developing and often extended periods of extreme dryness, followed by sudden water availability. These are called desiccation-tolerant or resurrection plants. An example is Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, an endemic

relict of the tertiary period. It may survive long dry periods between wet spells, passing quickly from anabiosis to full biological activity in less than 8–10 h if a favorable water balance in the soil is reestablished (Augusti et al. 2001, Quartacci et al. 2002, Sgherri et al. 2004, VeljovicJovanovic et al. 2006). The cellular mechanisms by which such rapid and ecologically beneficial changes in cellular activity are

Abbreviations – CBB, Coomassie brilliant blue; CGA, chlorogenic acid; DETC, diethyldithiocarbamate; DOPA, 3,4dihydroxyphenylalanine; HRP, horseradish peroxidase; IEF, isoelectrofocusing; POD, peroxidase; PPO, polyphenol oxidase; ROS, reactive oxygen species; RWC, relative water content.

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achieved in resurrection plants are not well understood, despite much study (e.g. Bartels and Salamini, 2001, Scott 2000). R. serbica has the ability to maintain cell integrity, preserving its plasma membrane lipid composition (Quartacci et al. 2002), which enables photosynthetic activity to be fully restored following re-watering (Augusti et al. 2001). A common feature of plants exposed to drought is the substantially increased release of reactive oxygen species (ROS), which are widely accepted to disrupt metabolism (Navari-Izzo and Rascio 1999) and lead to cell and tissue damage with severe dehydration in non-resurrection plants. An essential part of the adaptation of desiccation-tolerant plants is reduction of metabolic activities in a regulated manner, slowing down ROS production and inducing high antioxidant activity (Navari-Izzo et al. 1997, Sgherri et al. 1994a, 1994b, 2004). Ascorbate and glutathione are considered to be the main antioxidants in plants because of the direct removal of oxygen radicals by their involvement in the ascorbate–glutathione cycle. However, the antioxidant role of phenolics cannot be neglected (Sgherri et al. 2004). In R. serbica, the content of phenolic acids is unusually large in comparison with other plants (Booker and Miller 1998, Sgherri et al. 2004). Two enzymes, peroxidase (POD; EC and polyphenol oxidases (PPO; EC, may oxidize phenolics either by H2O2 or O2 and thus take part in the regulation of phenolic and polyphenolic concentrations in plants. PPO is a copper-containing protein that catalyzes two different reactions using molecular oxygen: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity). PPO is widely distributed in the plant kingdom, its role being mostly related to either cereal and fruit browning (Martinez and Whitaker 1995) or lignification (Bao et al. 1993, Savidge and UdagamaRandeniya 1992). However, the role of PPO in the protection against drought-induced damage in resurrection plants is unknown. Recent studies have indicated that phenol oxidizing enzymes may participate in the response to the defense reactions against various abiotic stresses including drought (Lee et al. 2007, Sofo et al. 2005, Wang et al. 2005). However, the results concerning droughtinduced changes in PPO activity are ambiguous and varied among crop species and conditions (e.g. Fazeli et al. 2007, Thipyapong et al. 2004, 2007), so the physiological role of PPO in plants during drought and recovery remains to be clarified. To our knowledge, PPO activity has not been investigated in resurrection plants so far, either for a role in dehydration or rehydration. The objective of this study was to characterize partially purified PPO in leaves of different water content in the resurrection plant, R. serbica. We hypothesize that changes in PPO activity and in the content 408

of PPO substrates and products during desiccation and rehydration may be part of the adaptation mechanism of these plants to severe drought conditions.

Materials and methods Plant material Specimens of the desiccation-tolerant plant Ramonda serbica Panc. & Petrov. were collected from their natural habitat in a gorge near the city of Nisˇ in south-eastern Serbia. Plants, collected with the attached layers of soil, were allowed to acclimatize for 4 weeks under full watering until the start of the experiments. One set of plants from three independent experiments was dehydrated by withholding water for 3 weeks at room temperature and ambient photoperiod. After this, rehydration was started by spraying the plants every 2 h with water to simulate rainfall, keeping the soil damp. Three samples were collected from fully hydrated leaves, desiccated leaves and during the first 2 days of rehydration until they become hydrated (relative water content, RWC – 91%). Another set of plants was watered daily during the whole experimental period. All measurements were carried out on mature and fully expanded leaves comparable in size and collected from the middle of the rosette. Relative water content Mature and fully expanded leaves, comparable in size (three replicates each from three independent experiments), were selected from the middle of rosettes of fully hydrated plants, dehydrated plants and during the 2 days of rehydration. Fresh, turgid and dry weights (DWs) of leaves were determined and RWC was calculated as follows: [RWC ¼ (fresh weight 2 DW)/(turgid weight 2 DW)  100]. Turgid weights were measured after rehydration in water for 24 h at 20C in the dark; DWs were obtained after the leaves had been dried for 36 h at 80C. Protein extraction and measurements of POD and PPO activity For enzyme analysis, leaves taken from the same plants as those used for RWC determination were weighed and frozen in liquid nitrogen. Plant material was crushed into powder in a mortar containing liquid N2 and extracted in 100 mM K-phosphate buffer (pH 6.5), 10 mM ascorbic acid, 2 mM phenylmethylsulfonyl fluoride and 2 mM EDTA with the addition of 5% (w/v) polyvinylpyrrolidine. The homogenate was centrifuged at 10 000 g for 15 min at 4C. The activity of PPO was determined either Physiol. Plant. 132, 2008

spectrophotometrically, as the initial rate of change in absorbance at 410 and 540 nm for chlorogenic acid (CGA) (4 mM) and 3,4-dihydroxyphenylalanine (DOPA) (25 mM), respectively, or polarographically by measuring oxygen uptake. Oxidation was followed in a Clark-type oxygen electrode (Hansatech, King’s Lynn, UK) in 100 mM K-phosphate buffer (pH 5.5) and 50 ml extract at 30C. The reaction was started by adding reductant (4 mM CGA, 25 mM DOPA or 10 mM methyl catechol) and the initial rate of oxygen uptake was determined. For estimation of apparent Km values, different concentrations of reducing substrates were used and apparent Km was calculated from Hanes plots for all substrates. For POD activity, pyrogallol (A430; e ¼ 2.47 mM21 cm21) was used as the hydrogen donor. The reaction mixture consisted of an aliquot of extract and 3.3 mM H2O2 in 100 mM K-phosphate buffer (pH 6.5) with 30 mM pyrogallol. Specific enzyme activity was estimated on a protein basis. The homogenate was centrifuged at 10 000 g for 15 min at 4C. The pellet of a crude leaf homogenate was washed three times with 100 mM K-phosphate buffer (pH 6.5). The ionically bound fraction was extracted from the washed pellet by incubation in NaC1 (1 M, 1 h, 4C). Thereafter, the pellet was washed three times with 100 mM K-phosphate buffer (pH 6.5). Finally, a covalently bound fraction was extracted from the washed and salt-extracted pellet by incubation (24 h, 4C) in the enzyme medium (0.5% pectinase and 2.5% cellulase). Protein content was measured according to the method of Bradford (1976). Electrophoresis Proteins were separated by native PAGE and isoelectrofocusing (IEF) to determine POD and PPO isoforms. Native electrophoresis was performed on 5% stacking and 10% running gel, with a reservoir buffer consisting of 0.025 M Tris and 0.192 M glycine (pH 8.3), at 24 mA for 120 min. SDS-PAGE was performed on 12% running gel according to Wang and Constabel (2003). Samples (15 mg of proteins) were diluted in loading buffer to final concentrations of 62.5 mM Tris–HCl, 0.1% (w/v) SDS, 10% (w/v) glycerol and 0.002% (w/v) bromophenol blue. Samples were loaded onto the gels without prior heat treatment. Following electrophoresis, PPO activity was visualized by staining the gels with 25 mM DL-3,4dihydroxyphenylalanine (DL-DOPA) in 10 mM sodium phosphate buffer (pH 6.0) containing 0.15% (w/v) SDS and 470 units ml21 catalase. Gels were stained until no further increase in color was apparently visible, usually within 30 min. Molecular mass standards (Bio-Rad, Munich, Germany) were visualized after staining with Coomassie brilliant blue (CBB) (0.1% CBB, 50% methPhysiol. Plant. 132, 2008

anol and 10% acetic acid). To detect POD activity, the gel was incubated with 10% 4-chloro-m-naphthol and 0.03% H2O2 in 100 mM potassium phosphate buffer (pH 6.5) after electrophoresis. IEF was carried out in 7.5% polyacrylamide gel with 3% ampholyte on a pH gradient from 3 to 9. Markers for IEF of pI range 3.6–9.3 were purchased from Sigma (Mannheim, Germany) (IEF-M1A). The amount of total protein applied to each well was 25 mg for native electrophoresis and 10 mg for IEF. The PPO activity was visualized by staining the gels with 25 mM DOPA in 10 mM sodium phosphate buffer (pH 6.0) containing 0.15% (w/v) SDS and 470 units ml21 catalase. Relative band intensities were estimated by measuring density using IMAGE MASTER TOTALAB v1.11. Phenolics determination Phenolic acids were determined as reported previously (Sgherri et al. 2004). Phenolic acids were extracted from turgid and dry leaves with 50% methanol containing 1% HCl under continuous stirring at room temperature. After centrifugation at 12 000 g for 15 min, the supernatant was collected and the extraction was repeated three times on the pellet. Methanolic extracts were collected, vacuum dried and resuspended in 80% methanol. Before analysis, samples were passed through a Sartorius filter (Minisart 0.45 mm) to remove any suspended material. A measure of total phenolics was obtained by recording A280 before and after addition of Polyclar AT (BDH Chemicals Ltd, Poole, UK) to the extract (1:10 w/v) as reported by Sgherri et al. (2004). The calculations were performed using the absorbance, obtained by the difference between the two readings, and a calibration curve for total phenolics prepared with gallic acid as standard. Statistical analysis The results are the means of three replicates from four independent experiments. Comparisons among the means to determine significant differences (P  0.05) were performed with the Mann–Whitney U test.

Results Comparison of POD and PPO activities Both CGA and pyrogallol may be oxidized in the POD reaction by class III POD and both are common substrates for PPO too. Comparisons of fully hydrated (RWC ¼ 95%) and desiccated leaves (RWC ¼ 2%) for oxidizing and peroxidizing activity in the presence of either CGA or pyrogallol showed significant differences 409

(Fig. 1). While addition of H2O2 to the reaction mixture with leaf extract and pyrogallol enhanced the generation of products at a similar rate in fully hydrated and desiccated leaves (Fig. 1B), addition of H2O2 in the presence of CGA did not increase absorbance at 410 nm (Fig. 1A). Neither fully hydrated nor desiccated R. serbica leaf extract oxidized CGA with H2O2. In contrast, polarographic measurements of O2 consumption confirmed that the absorbance increase at 410 nm was due to oxidation of CGA by molecular oxygen (Fig. 1A, inset). Comparable Vmax values were determined for CGA and methyl catechol, while polyphenol oxidizing activity was very low in the presence of DOPA (Fig. 2). Calculation of Km revealed that an enzyme with polyphenol oxidizing activity had the highest affinity for methyl catechol (apparent Km ¼ 0.4 mM), while similar apparent Km values were determined for CGA (1.8 mM) and DOPA (2.1 mM) (Fig. 2). Estimated apparent Km for molecular oxygen for CGAdependent PPO activity (48 mM) showed high affinity of PPO for oxygen (Fig. 2, inset). We also measured the content of total phenolic acids in turgid and dry leaves. During the drought period of 2 weeks, the content of phenolics decreased from 2.71 mg g21 DW in turgid leaves to 0.95 mg g21 DW in dry leaves. The sensitivity of POD and PPO to various inhibitors was comparatively analyzed with leaf extract to distinguish between the two enzymes (Table 1). The specific POD inhibitors, KCN, salicylic hydroxamic acid and DTT, inhibited PPO and POD activities to a similar extent. However, a specific inhibitor of Cucontaining enzymes, diethyldithiocarbamate (DETC), was the most selective for PPO, while NaN3 at 3 mM inhibited POD activity by 88% but had no effect on PPO activity (Table 1).

Fig. 2. Specific rate of PPO-induced oxidation of different phenolics measured as oxygen consumption. The reaction was started by addition of reducing substrate CGA (s), methyl catechol (h) and DOPA (n, right y-axis). Measurements were preformed at 30C in 100 mM K-phosphate buffer pH 5.5 for CGA-dependent PPO and pH 6.5 for methyl catechol and DOPA. Values are means (SD) for three or four leaves collected in each of the three independent experiments. Km values were calculated for each substrate after linearizing average values using Hanes plots. The inset shows CGA-dependent PPO activity determined at different concentrations of O2. Oxidation was followed in a Clark-type oxygen electrode.

To resolve PPO from POD isoforms, we performed PAGE analysis of R. serbica extract in control plants and commercial horseradish peroxidase (HRP, type II, from Sigma) in the presence of the specific inhibitor of PPO, DETC (Fig. 3). Two band groups of distinctive molecular weight and substrate specificity were determined by native PAGE: one with high molecular weight and PPO activity and the other with lower molecular weight and POD activity (Fig. 3). In contrast to the bands of HRP and R. serbica POD, which did not show PPO activity with DOPA, the band with PPO

Fig. 1. Effect of desiccation on oxidizing and peroxidizing activities of Ramonda serbica leaf extracts. Extract-induced (turgid – TL, desiccated – DL) absorbance increase at 410 nm for CGA (A) and at 430 nm for pyrogallol (B) was followed before and after addition of 0.3 mM H2O2 (marked by arrows) in Pi-buffer, pH 6.5. Inset shows CGA-dependent oxygen consumption by extracts of turgid and dry leaf measured polarographically in the same reaction mixture as used for spectrophotometric measurement in absence of H2O2.


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Table 1. Effects of different inhibitors on pyrogallol-dependent POD and CGA oxidase activity of leaf extracts of Ramonda serbica. Inhibition was expressed as a percentage of the control enzyme activity determined by measuring the rate of absorbance increase at 430 nm for POD or the rate of oxygen consumption polarographically in the case of PPO. % Inhibition

Inhibitors DETC KCN Salicylic hydroxycinnamic acid Na3N DTT Phenylhydrazine Phenylhydrazine

Concentration (mM)

Pyrogalloldependent POD activity

CGAdependent PPO activity

0.2 1.0 3.0

34  9 90  3 91  7

91  3 95  2 100

3.0 1.0 0.2 1.0

88  20 100 61 25  4

82 100 82  7 92  2

activity in R. serbica extract disappeared after treatment with DETC, confirming that its oxidizing ability cannot be assigned to POD. Most of the enzyme activity (79%) was found in the soluble fraction of leaf extract, but about 20% of the total activity was ionically or covalently bound to cell constituents (Table 2). PPO activity was also highly sensitive to phenylhydrazine (Table 1), with more than 80% of activity inhibited with 0.2 mM phenylhydrazine in both fully hydrated and desiccated leaves. The inhibition increased 10 s after reaction was started by addition of CGA to the same extent in both cases.

Fig. 3. Specific inhibition of PPO activity in Ramonda serbica leaves with DETC. Leaf extracts of R. serbica (lane 1) and HRP (lane 2) were compared for PPO and POD sensitivity with 1 mM DETC by incubating the gel with inhibitor for 15 min. Native PAGE was prepared as described in the Materials and methods without addition of SDS. For PPO isoforms, gels were stained with DL-DOPA and for visualization of POD isoforms with 4-chloronaphthol. Arrows indicate POD and PPO isoforms.

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PPO isoforms induced by desiccation Using specific SDS electrophoresis that maintained PPO active (Wang and Constabel 2003), we identified a small molecular form of PPO with an approximate molecular weight of 35 kDa (Fig. 4A). Over the 2-week period of water deprivation, the RWC of leaves gradually decreased from 95 to 2%. During dehydration, the amount of this PPO isoform in leaf increased four-fold as indicated by band density. Separation of PPO isoforms by their pI values revealed one isoform with pI 4.8 in both fully hydrated and desiccated leaves, which was increased by 250% in the latter. IEF revealed three additional anionic isoforms with pI 5.1, 5.3 and 5.6 and three neutral isoforms with pI from 6.8 to 7.4 in desiccated leaves only (Fig. 4B). After 1 week at 2% water content, the leaves were rehydrated. During the first few hours of rehydration, PPO was inactivated and then activity increased (Fig. 5A). The intensity of the 35 kDa PPO isoform showed differences with time depending on leaf water status. Thus, in the first few hours after rewatering desiccated leaves, the intensity of the band decreased greatly but then recovered within 24 h to an intensity similar to that for desiccated leaves, i.e. four-fold higher than the band density of turgid leaves, which had not experienced water loss. In addition, the intensity of the pI 4.8 isoform increased and new bands with pI from 5 to 5.6, 6.8 and 7.4 reappeared after 48 h of rehydration (Fig. 5B).

Discussion We have shown here that R. serbica leaves contain high PPO activity, which is several fold higher in desiccated leaves (Figs 1, 4 and 5). Water loss from R. serbica resulted in a three-fold decrease in total phenolic acid content in desiccated leaves, in agreement with the data of Sgherri et al. (2004). This indicates that phenolic oxidation plays an important role in the adaptation mechanism to water deficit. Evidence is accumulating to support the idea that phenolics also play a role in defending the plant against various biotic and abiotic stresses (Munne-Bosch and Alegre 2004, Pourcel et al. 2006, Treutter 2006). Phenolic acids are common substrates for class III POD, but in agreement with previous results (Veljovic-Jovanovic et al. 2006), we did not find significant changes in POD activity in desiccated leaves (Fig. 1). Instead, there was a large increase in CGAdependent oxidase activity (Fig. 1). In desiccated leaves, four-fold higher PPO activity was induced, a result that points to PPO is an important regulator of phenolic content in R. serbica. When non-denaturing SDS-PAGE was used, one PPO isoform with an apparent molecular weight of 35 kDa 411

Table 2. Distribution of PPO and POD activity among leaf fractions: soluble, obtained by extraction with Pi-buffer pH 6.5; ionically bound, obtained by extraction of proteins from leaf pellets with 1 M KCl; and the protein fraction that stayed covalently bound to leaf tissue. The specific activity of DOPA- and CGA-dependent PPO and pyrogallol (PG)-dependent POD was expressed on a protein basis and the total enzyme activities in relation to the fresh weight (FW) of turgid mature leaves. Specific activity

Total activity


PG-POD (mmol mg21 protein min21)

DOPA-PPO (DA mg21 protein min21)

CGA-PPO (DA mg21 protein min21)

PG-POD (mmol g21 FW)



Soluble Ionically bound Covalently bound

3.44  0.19 2.76  0.35 —

21  2 5  0.3 —

49  2 32  2 —

44  3 3.7  0.1 1.7

272  15 6.7  0.1 17  1

641  61 43  3 118  6

(Fig. 5A) was obtained, which is similar to the molecular weight reported elsewhere (Kihara et al. 2005). The different enzyme mobility obtained with native PAGE (Fig. 3) may be explained by the presence of dimers or tetramers in situ, as reported by Wang et al. (2005). Most of the enzymes from leaf extracts of R. serbica appear to be soluble (Table 2), probably located in chloroplasts (Golbeck and Cammarata 1981), where it might act as an oxygen buffer or scavenger in the lumen of thylakoids (Bar-Nun and Mayer 1983). In turgid leaves, IEF also revealed two PPO isoforms of the same pI (4.5) as found for POD anionic isoforms (Veljovic-Jovanovic et al. 2006). All other PPO isoforms found in dehydrated leaves had pI values distinct from those of POD isoforms (Fig. 4B).

Fig. 4. Non-denaturating SDS-PAGE (A) and IEF (B) electrophoresis of soluble proteins stained for PPO activity in Ramonda serbica leaves of turgid (lane 1, RWC 95%) and desiccated (lane 2, RWC 2%) plants. IEF was performed in a pH gradient of 3–9. Arrows indicate different PPO isoforms with different pI values. An amount of 50 mmol of total proteins was applied to each well.


Fig. 5. Non-denaturating SDS-PAGE (A) and IEF (B) electrophoresis of soluble proteins stained for PPO activity in Ramonda serbica leaves of turgid and dehydrated plants and during rehydration. Rehydration lasted 48 h and the scale indicates the time after the beginning of watering. Arrows point to PPO isoforms with different pI values. An amount of 50 mg of total proteins was applied to each well. The table below ‘A’ indicates the rate of oxygen consumption by PPO in the presence of 4 mM CGA and table below ‘B’ shows RWC values of leaves taken at marked times from the same plants used for enzyme analysis. TL, turgid; DL, desiccated.

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Comparative analysis of the effects of inhibitors on PPO and POD activities (Fig. 3, Table 1) provided means to differentiated POD and PPO, despite the similar pI (4.5) of the most abundant isoforms in turgid leaves. While no POD activity was measured in the presence of azide (1 mM), we found PPO activity to be resistant to treatment with NaN3 but highly sensitive to the Cu-chelating agent, DETC. Although azide is considered to be an inhibitor of PPO, because it can form complexes with many copper enzymes (Gromov et al. 1999), it has also been reported to have an activating role (Shi et al. 2002). Inhibition of PPO by phenylhydrazine (Table 1) classifies this oxidase in the group of o-diphenol oxidases rather than the laccase-type oxidases (Bligny and Douce 1983, Mayer 1987). No oxidizing activity was found with 2,2#azinobis-(3-ethylbenzothiazoline)-6-sulfonic (data not shown), which is a commonly used substrate to test the laccase type of PPO found in fungi (Nagai et al. 2003). PPO activity of R. serbica showed the highest rates of oxidation with CGA and methyl catechol, although the latter is the preferred substrate (Fig. 2). CGA is considered to be the endogenous reductant for class III PODs, which use phenolics as preferential electron donors, as shown for PODs originating from various species (Kukavica and Veljovic-Jovanovic 2004, Takahama et al. 1999). However, we found that the oxidation of CGA by molecular oxygen catalyzed by PPO is the preferred reaction in leaf extracts of R. serbica (Fig. 1A, inset). By addition of either H2O2 (Fig. 1) or catalase (data not shown) to the reaction mixture, we confirmed that POD did not contribute to the CGA-dependent oxidation induced by leaf extract. PPO could also oxidize DOPA, although at much lower rates (Fig. 2, Table 1). Compared with our results, the same Km for methyl catechol was obtained for PPO from poplar but a much higher Km for CGA and DOPA (Wang and Constabel 2003). PODs may also contribute to enzymatic browning by oxidizing hydroxycinnamic acid derivatives and flavans (Takahama and Oniki 2000), i.e. the main phenolic structures implicated in enzymatic browning. Products of flavonoid and phenolics oxidation may have an even stronger scavenging effect on ROS than their respective monomers (Kono et al. 1997, Pourcel et al. 2006). The oxidation products of diphenolase activity, o-quinones, may have also a beneficial role in phytochemical defense of plants (Haruta et al. 2001, Rahman and Punja 2005). The large amounts of PPO induced by drought have been shown in numerous crop species (English-Loeb et al. 1997, Jiang et al. 2007, Shivishankar 1988, Thipyapong et al. 2007) that strongly implies an important role in adaptation strategy to drought conditions. The effort on improving the adaptation of crop plants to drought conditions has been increasing (Thipyapong et al. 2004, Physiol. Plant. 132, 2008

2007, Wang et al. 2005); yet, the mechanism of PPO contribution to defense processes is ambiguous scarce and more comprehensive study has to be carried out in future. Thipyapong et al. (2007) proposed the mechanism by which electrophilic products of PPO reaction induced cell death in older leaves and abscission zones that would improve redistribution of nutrients to younger tissue of tomato plants. In resurrection plants, however, leaves do not die during long period of desiccation, but reestablish all biological processes within 1 day on rehydration. We propose a role of PPO in resurrection plants in the preservation of proteins and cell constituents during long periods of drought. A similar function for PPO has been shown for postharvest processes in fruits (Nicolas et al. 1994, Sullivan and Hatfield 2006). Resurrection plants are rich in flavonoids, which, besides CGA, are preferable substrates for PPO (Pourcel et al. 2006). In resurrection plants, both functions are of great importance as dried leaves are exposed to pathogens, insects and proteases and other degradative processes (Chazarra et al. 2001). In the apoplast, this can be one of the mechanisms involved in defense against pathogen attack (Li and Steffens 2002, Melo et al. 2006) and wounding (Mayer and Harel 1979). It is widely accepted that the most dramatic period regarding cellular oxidative injury is when plant rehydration begins (Navari-Izzo and Rascio, 1999, Sgherri et al. 1994a, 1994b). We suggest that this occurs because of the inability of cellular enzymatic antioxidative systems to buffer water that initiates abrupt oxidative changes in the still disrupted cell structure. Rehydration brought about short-term disappearance of the PPO isoforms (with pI from 5 to 7.4, Fig. 5B) present in desiccated leaves, which were then reinduced within 1 day during rehydration. This observation (Fig. 5) is in accordance with the transient decrease of SOD, APX and POD activities occurring in the first few hours of rehydration (Veljovic-Jovanovic et al. 2006). The transient inactivation of the antioxidative enzymes might be the consequence of uncontrolled radical chain reactions due to small increase in RWC leading to oxidation of the proteins (Davies et al. 1987). However, while the activities of ascorbate peroxidase (APX), superoxide dismutase (SOD) and POD returned to the initial values in turgid leaves or stayed at lower level, PPO activity increased to the highest values that were measured in desiccated leaves. Further investigation on PPO transcripts and enzyme activity during rehydration in leaves that had been experienced desiccation are needed to elucidate its role in adaptation strategy to drought conditions. A transient increase in phenolics (hydroxycinnamates), during PPO inhibition in the first hours, might be beneficial because of their chelating ability, which would prevent lipid peroxidation (Quartacci et al. 413

2002) induced either by Cu(II) or Fe(III) (Morel et al. 1997, Psotova et al. 2003, Sakihama et al. 2002), thus explaining their antioxidant capacity. Results imply the important role for PPO oxidation substrates as antioxidants in rehydration period.

Conclusions Our results show that the enzyme extracted from R. serbica leaves has properties (i.e. stability to SDS, solubility, independence toward H2O2, sensitivity to DETC, substrate specificity and an apparent molecular weight of 35 kDa) that are characteristic of catechol oxidase (EC The large amount of PPO induced by drought strongly implies a protective role, probably in the preservation of proteins and cell constituents during long periods of drought, as well as in ROS scavenging by polyphenolics, which are PPO oxidation products. However, direct cooperation between POD and PPO in the browning reaction occurring in situ may not be excluded. Oxidative stress caused by transient inactivation of PPO together with other antioxidant enzymes in R. serbica in the first hours after rehydration might be overcome by an abrupt increase in the content of phenolics that would prevent lipid peroxidation by their antioxidant and chelating ability. Results indicate the responsiveness of PPO to dehydration of R. serbica may be a part of strategy to cope with multitude of stress that often occurs simultaneously, especially during long period of desiccation. Acknowledgements – This work is financed by the Ministry of Science of the Republic of Serbia (Project No. 143020B) and is the result of cooperation between the University of Pisa and the University of Belgrade.

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