JXB Advance Access originally published online on May 19, 2006
Journal of Experimental Botany 2006 57(8):1759-1768; doi:10.1093/jxb/erl007
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RESEARCH PAPER |
Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica
1Center for Multidisciplinary Studies, Belgrade University, Kneza Vi
eslava 1a, 11030 Belgrade, Serbia and Montenegro
2Faculty of Biology, Belgrade University, Takovska, 11000 Belgrade, Serbia and Montenegro
3Dipartimento di Chimica e Biotecnologie Agrarie, Università degli Studi di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy
*To whom correspondence should be addressed. E-mail: fnavari{at}agr.unipi.it
Received 1 March 2006; Accepted 29 March 2006
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Abstract |
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Ramonda sp. (Gesneriaceae) is an endemic and relic plant in a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. Senescence- and drought-related changes in the activity of peroxidase (POD; EC 1.11.1.7 [EC] ), ascorbate peroxidase (EC 1.11.1.11 [EC] ), and superoxide dismutase (SOD; EC 1.15.1.1 [EC] ) were determined in leaves of different age and relative water content. The results indicate that different POD isoforms were stimulated during senescence and dehydration. Two of the soluble POD isoforms were anionic with pI 4.5, and two were cationic with pI 9.3 and 9.0. The activity of ascorbate peroxidase remained unchanged either by drought or senescence. For the first time, SOD isoforms have now been determined in this resurrection plant. Several SOD isoforms, all of the Mn type, were found to be anionic with pI 4 and a few others had pI from 5 to 6, while one band of FeSOD with a lower molecular weight was neutral. Rehydration brought about a remarkable decrease over the first hour in the activity of all the antioxidant enzymes examined but activity recovered 1 d after rehydration. The results confirmed that dehydration and senescence caused disturbance in the redox homeostasis of Ramonda leaves, while inducing different POD isoforms. A physiological role of peroxidase reaction with hydroxycinnamic acids in conservation and protection of cellular constituents of desiccated Ramonda leaves is suggested.
Key words: Desiccation, peroxidase, Ramonda, senescence, superoxide dismutase
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Introduction |
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Water deficit, like other abiotic and biotic stresses in plants, may disturb the redox homeostasis and may even lead to oxidative stress under severe conditions (Winston, 1990; Price and Hendry, 1991; Quartacci and Navari-Izzo, 1992; Vertucci and Farrant, 1995; Navari-Izzo et al., 1995). Peroxidase (POD) and superoxide dismutase (SOD) constitute the first line of defence against reactive oxygen species (ROS), and changes in their activity and amounts have been identified as an indicator of a redox status change under drought conditions in homoiohydric plants (Moran et al., 1994; Schwanz and Polle, 2001). Under prolonged harsh environmental conditions, most plants that are homoiohydric or desiccation intolerant get irreversibly damaged, which leads to early senescence and sudden death. Less than 1% of flowering plants belong to a group known as ‘resurrection’ plants (Porembski and Barthlott, 2001), which are able to survive by maintaining metabolic functions in an almost completely dehydrated state, and then to recover their activity readily upon rehydration (Gaff, 1989). The capacity to recover, based on an alteration in gene expression that becomes evident upon rehydration, is especially strong and extremely rapid in poikilohydric desiccation-tolerant plants. Investigation of this unique adaptation mechanism to water deficit, developed through dehydration and recovery of cell constituents after a state of anabiosis, may give new insights into the process of water stress in plants or even help improve drought resistance in crops. Over the years it has also become clear that free-radical scavenging systems are important components in the mechanisms of drought and desiccation tolerance (Navari-Izzo et al., 1997; Ingram and Bartels, 1996; Noctor et al., 2002). Ramonda serbica Panc. (Gesneriaceae) belongs to a very small group of homoiochlorophyllous and poikilohydric angiosperms of the northern hemisphere that includes five or six species (families Gesneriaceae and Cyperacea). Ramonda sp. is an endemic and relic plant of the tertiary period originating from the Balkan peninsula, and is a resurrection, perennial, herbaceous, and shade-adapted species. Studies of the metabolism of Ramonda sp. plants under dehydration and subsequent rehydration (Augusti et al., 2001; Quartacci et al., 2002) have indicated a role of oxidative processes in impairment of photosynthesis and plasma membrane structures. Despite many studies of the distribution and activity of isoforms of antioxidative enzymes in other plants (Polle et al., 1994; Bernards et al., 1999; Schwanz and Polle, 2001), no analyses have so far been made of POD and SOD isoform profiles in Ramonda sp. plants. Accumulation of ROS during senescence (Leshem, 1981; Thomson et al., 1987; Mahalingam and Fedoroff, 2003) has been shown to accompany programmed cell death, implicating the impairment of cellular antioxidant defence (Kar and Feierabend, 1984; Jiménez et al., 1998). Although changes in the activities of ascorbate peroxidase (APX), POD, SOD, and catalase, during senescence of homoiohydric plants have been reported (Pastori and del Río, 1994; del Río et al., 1998; Prochazkova et al., 2001; Kukavica and Veljovic-Jovanovic, 2004), there is no clear evidence as yet of a physiological role of these enzymes in senescence. A correlation between desiccation- and senescence-induced changes in antioxidative metabolism has been reported, showing that some desiccation-tolerant plants may lose tolerance with ageing (Gaff and Giess, 1986; Gaff, 1989). For the first time, senescence-related changes in the antioxidative metabolism in one of the resurrection plants have been investigated. The objective of this study was to analyse peroxidase and SOD changes in the resurrection plant Ramonda serbica, an attractive model-system plant, in senescence and, upon dehydration and rehydration, to evaluate similarities in antioxidative responses induced under these conditions. In accordance with the widespread classification of peroxidases, the superfamily is subdivided into three classes based on sequence alignments and biological origin (Welinder, 1992). Two classes of peroxidase, i.e. ascorbate peroxidase (EC 1.11.1.11 [EC] ) that belongs to class I and glycosylated unspecific peroxidase (POD; EC 1.11.1.7 [EC] ) belonging to class III, were analysed. It remains to be clarified in future studies whether there is a unique mechanism underlying antioxidative defence, or are there some basic properties conferring such ‘broad spectrum’ tolerance to oxidative stress.
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Plant material
Specimens of the desiccation-tolerant plant Ramonda serbica Pan. & Petrov. were collected from their natural habitat in a gorge near the city of Ni
in south-eastern Serbia. Plants were harvested together with the attached layers of soil. After collection, the plants were acclimated for 4 weeks under full watering until the beginning of the experiments. For the senescence study, leaves of different ages, comparable in size, were collected from four plants according to their position in the rosette: the young leaves which were also the smallest in size were taken from the central part, the mature and fully expanded ones from the middle, and the senescent yellowish ones from the peripheral part of the rosette (Fig. 1). After taking samples for the senescence study, the plants were subjected to drought and relative water content (RWC) was measured in leaves sampled from the same plants (Fig. 2). The plants were dehydrated for 2 weeks by withholding water, and were kept under room temperature and ambient photoperiod. After this period, rehydration was induced by spraying the plants every 2 h with water to simulate rainfall and to keep the soil damp. The samples were collected during dehydration and over the first 3 d of rehydration.
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Relative water content
Measurements of RWC were taken at regular intervals (every other or third day during dehydration, and at 6 h intervals during the first day of rehydration, and on the second and third days of rehydration) according to a reported procedure (Sgherri et al., 1994a). Mature and fully expanded leaves from the middle part of the rosette, comparable in size, were selected for the analyses. RWC of leaves was calculated according to formula: 100x[(fresh weight–dry weight)/(saturated weight–dry weight)].
Protein extraction and measurements of POD activity
For enzyme analysis, leaves taken from the same plant as the one used for RWC determination were weighed and frozen in liquid N2. 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 PMSF (phenylmethylsulphonyl fluoride), and 2 mM EDTA with the addition of 5% (w/v) insoluble polyvinylpyrrolidine. The homogenate was centrifuged at 10 000 g for 15 min at 4 °C. To determine peroxidase activity, pyrogallol (A430; 
=2.7 mM–1 cm–1), guaiacol (A470; =2.6 mM–1 cm–1), caffeic acid (A450), and ferulic acid (A356) were used as hydrogen donors. In the case of hydroxycinnamates, the rate of peroxidase reaction was measured as an absorbance increase. The Km values were calculated from Hanes plots for all substrates. The reaction mixture consisted of an aliquot of extract diluted 300-fold and 3.3 mM H2O2 in 100 mM K-phosphate buffer (pH 6.5), with 30 mM pyrogallol or a different concentration of reducing substrates. To measure APX activity, absorbance decrease at 290 nm (=2.8 mM–1 cm–1) was monitored in a reaction mixture consisting of 0.3 mM ascorbate, 0.1 mM H2O2, and 50 µl extract in 50 mM K-phosphate (pH 7.2). Estimation of specific enzyme activity was done on a protein basis. Protein content was measured according to Bradford (1976).
Electrophoresis
SDS-PAGE was performed on 12% running gel. Samples (15 µg 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, 100 mM DTT, and 0.002% (w/v) bromophenol blue. After heat treatment for 30 min at 100 °C, samples were loaded onto the gels. Protein bands were visualized after staining with CBB (0.1% CBB, 50% methanol, 10% acetic acid). Molecular mass standards (Bio-Rad) were used. Proteins were separated by native PAGE and isoelectrofocusing (IEF) to determine POD and SOD 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 Gly (pH 8.3), at 24 mA for 120 min. IEF was carried out in 7.5% polyacrylamide gel with 3% ampholyte on a pH gradient of 3–9. Markers for IEF of the pI range 3.6–9.3 were purchased from Sigma (IEF-M1A). The amount of total protein applied to each well was 50 µg for native electrophoresis and 25 µg for IEF. To determine POD activity, the gel was incubated with 10% 4-chloro-
-naphthol and 0.03% H2O2 in 100 mM K-phosphate buffer (pH 6.5). Determination of SOD activity on the gels was performed according to Beauchamp and Fridovich (1971). After incubation in reaction mixture (0.1 M EDTA, 0.098 mM nitroblue tetrazolium, 0.03 mM riboflavin, and 2 mM TEMED in K-phosphate buffer, pH 7.8) for 30 min in the dark, the gel was washed in distilled water and illuminated. Different SOD isoenzymes (CuZn, Mn, and FeSOD) were identified by preincubation with either 5 mM KCN or 5 mM H2O2 before staining. Relative band intensities were estimated, measuring density with Total Lab.
Statistical analysis
Data were analysed with Mann–Whitney U test. Comparison tests were used to determine levels that differed significantly (P <0.05).
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Changes in soluble proteins and peroxidase
Throughout the dehydration–rehydration cycle, leaves of the fully hydrated control plants showed no significant differences in RWC or enzyme activities. For this reason the data relative to control plants represent the mean value of the control plants at each harvest time. The mean RWC decreased from 97% in fully hydrated leaves of control plants to 7% in the dried ones (Fig. 1). The rate of water loss was quite low over the first 3 d, RWC decreasing only by about 3% each day. Soon the rate of water loss increased so that, after the next few days of withholding water, the RWC was lowered to 58–60%, and in the following 5 d it decreased further down to 7% (Fig. 2). After 12 h of rehydration, which took place after 4 d, the leaves quickly regained RWC and recovered to control values after 70 h. Compared with mature turgid leaves (referred to as controls), the protein level in senescent turgid Ramonda leaves was found to be 20% lower, and in dehydrated mature leaves 40% and 20% lower (at 50% and 20% RWC, respectively) than in controls (Table 1). However, comparison of the electrophoretic patterns of denatured proteins from heat- and SDS-treated leaf extracts of senescent and dehydrated leaves revealed significant differences in the contents of peptides (Fig. 3). In senescent leaves all bands had rather low intensity and some of them were completely missing in comparison with young and mature leaves, while the bands of dehydrated leaves were more distinct and some were intensified. It was noticed that by measuring band density the intensity of the band with Mr 60 increased by 38–47%, the Mr 31 band increased by 69–87% and the one with Mr 14 increased by 80–86% in dehydrated leaves compared with mature turgid leaves. New bands with Mr 33 and 25 appeared upon dehydration (Fig. 3). To measure class III POD activity, pyrogallol as an electron donor, guaiacol and two hydroxycinnamic acids, i.e. caffeic and ferulic acids, were used. The highest specific activity was measured for guaiacol, and the lowest for ferulic acid (Fig. 4). Calculated Km (mM) values obtained from Hanes plots were 0.4 for caffeic acid, 1.9 for ferulic acid, 5.6 for guaiacol, and 5.4 for pyrogallol. Increase in the activity of POD, with pyrogallol as electron donor, was significant both in the senescent and dehydrated leaves (50% RWC) compared with mature and control leaves (Table 1). In leaves with 20% RWC, the specific POD activity was similar to the activity measured in control leaves. APX activity in young leaves measured half the activity in mature leaves and did not change during senescence or drought (Table 1). Separation of POD isoforms based on molecular weights showed differences in POD profiles between mature, young, and old leaves (Fig. 5). A form of high mobility that was present in young leaves disappeared in mature and old leaves, while a form of low mobility appeared only in old leaves. Dehydration of plants down to 50% and 20% RWC induced two additional isoforms with higher mobility than in mature leaves. Separation of POD isoforms by their pI values revealed two anionic isoforms with similar pI (4.5) and two cationic ones with pI of about 9.3–9.0 (Fig. 6). During ageing, anionic isoforms increased by 180% in mature and 280% in senescent leaves compared with young leaves, while drought had no influence on the amount of anionic isoforms. On the other hand, the intensity of cationic isoforms was 4-fold higher in the leaves of 50% RWC, and two new isoforms with pI 8.4 and 8.6 appeared. In the leaves with 20% RWC, two cationic isoforms (pI 9.3–9.0) were 3-fold intensified compared with turgid leaves. The anionic isoforms remained unchanged in leaves of different RWC (Fig. 6). Over the 2 week period of withholding water, leaves were gradually dehydrated from 95% to about 7% RWC (Fig. 2). Leaves with similar RWCs were grouped and the enzyme activity plotted against the RWC of the corresponding plant (Fig. 7). Specific POD activity increased significantly (from 8.7 µmol mg–1 protein min–1 in turgid leaves to 17.1 µmol mg–1 protein min–1 in the leaves of 57% RWC), and decreased gradually as dehydration progressed (Fig. 7B). POD activity varied greatly in the desiccated leaves due to different ages. APX activity was measured in fresh extracts of Ramonda leaves as a 50% decrease in the activity was detected after 2 h of storage on ice (data not shown). When specific APX activity was plotted against the RWC of leaves, it showed no significant changes even in desiccated leaves (Fig. 7A).
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0.05).A transient decrease in the activity of both APX and POD was followed by an increase on the first day of rehydration (Fig. 8). Whereas POD activity decreased again and did not rise to control values even after 70 h, APX reached an activity similar to control leaves after 10 h (Fig. 8).
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Changes in SOD isoforms during dehydration and rehydration
Both senescence and dehydration brought about a slight increase in the level of SOD, which is particularly evident on the IEF gel (Fig. 10). Using specific inhibitors, KCN and H2O2, several SOD isoforms were identified in Ramonda leaves, five of them being MnSOD and one of higher mobility FeSOD (Fig. 9). However, it was not possible to identify CuZnSOD in Ramonda leaves. IEF revealed numerous MnSOD anionic isoforms with pI from 6 to 5 and one with pI 4, while one band of FeSOD of smaller molecular weight was neutral. Interestingly, a high level of SOD activity was found in dried Ramonda leaves. SOD was inactivated during the initial hour of rehydration (Fig. 11). The recovery of SOD activity in Ramonda occurred within 24 h of rehydration.
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Discussion |
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The production of ROS such as the superoxide radical increases in senescence (del Río et al., 1998) and under drought stress (Navari-Izzo and Rascio, 1999; Alscher et al., 2002), leading to disturbance in the redox homeostasis and also playing a key role in redox signalling in leaves (Foyer and Noctor, 2003). Increases in POD and MnSOD activity in dehydrated and senescent Ramonda leaves (Figs 5, 7, 9; Table 1) can therefore be considered part of an antioxidative defence under such conditions. An increase in total peroxidase activity in Ramonda leaves during dehydration has been already reported (Sgherri et al., 2004). In the present experiment, this increase did not continue with decreasing RWC, but POD activity decreased to the values obtained in turgid mature leaves (Fig. 7). The enzymes examined were not inactivated in desiccated leaves but they stayed intact and conserved, being transiently inactivated only upon rewatering. The different result in dried leaves could lie in the higher (7%) RWC reached in the present experiment in comparison with the experiment of the previous authors, where the RWC attained was 4.2%. In addition, IEF analysis reveals that different isoforms were being induced in senescence and drought (Fig. 6). Dehydration induced cationic and POD isoforms with lower molecular weight, while anionic isoforms with pI 4.5 were induced in senescent leaves. One of the challenges associated with studying the peroxidases remains the determination of the physiological role of different isoforms. All efforts to assign a specific function to any particular peroxidase isoform (Goldberg et al., 1983; Polle et al., 1994; Bernands et al., 1999) is complicated by the broad spectrum of substrates and the ability of class III peroxidases (glycosylated unspecific peroxidases) to compensate the absence of a specific isoform with another. The present analysis of substrate specificity of Ramonda POD showed a similar affinity for phenols, i.e. guaiacol and pyrogallol (Km 5.6 and 5.9 mM), and higher affinity for caffeic and ferulic acids (0.4 mM and 1.9 mM, respectively). In Ramonda serbica the content of phenolic acids was unusually elevated in comparison with other plants (Booker and Milller, 1998), having a high level of chlorogenic, protocatechuic, and p-hydroxybenzoic acids (Sgherri et al., 2004). The decrease in total phenolic acids during dehydration suggests their important role in antioxidative defence. A protective role of the peroxidase/phenolic/ascorbate cycle (Takahama and Oniki, 1997; Takahama, 2004) operating in the vacuole and apoplast of Vicia faba against oxidative stress (Takahama and Oniki, 1997) may be assumed as a detoxification system of H2O2 in Ramonda subjected to dehydration.
While class III peroxidases, which use phenolics as preferential electron donors, participate in polymerization reactions in the cell wall, the metabolism of indolacetic acid or ethylene (De Gara, 2004), APX (class I peroxidase) uses ascorbate as the preferential electron donor and operates as a H2O2 scavenger within the cell. Although a decrease in the ascorbate content is often considered to be an indicator of senescence in many annual plants (Takahama et al., 1999; Dertinger et al., 2003) APX activity was shown not to change in senescent leaves, but rather to remain the same throughout the period of drought (Fig. 7; Table 1). However, contrasting results related to ascorbate and glutathione levels and the redox state need further investigation of ascorbate metabolism in this unique plant (Augusti et al., 2001; Sgherri et al., 2004). A simultaneous decrease in the levels of phenolics reported by Sgherri et al. (2004) for Ramonda leaves during dehydration and increase in POD activity suggests the role of the peroxidase/phenolics/ascorbate system in scavenging ROS and releasing oxidative stress during dehydration. An increase in the content of phenolic acids over the first hour of rewatering, in the initial hour of rehydration (Sgherri et al., 2004) may result from an intensified biosynthesis and decreased peroxidase activity (Fig. 8B). Rehydration also brought about a transient decrease in the activity of the other two antioxidant enzymes, SOD (Fig. 11) and APX (Fig. 8A), and an increase to higher activity in the following days, suggesting their role in alleviating oxidative stress during rehydration. Results confirm that the most dramatic period regarding cellular oxidative injury is when plant recovery begins (Sgherri et al., 1994a, b; Navari-Izzo and Rascio, 1999; Augusti et al., 2001). The leaf of Ramonda has several SOD isoforms, most of them being MnSODs and one of them FeSOD. FeSOD is an ancient form of SOD that is localized in the chloroplasts of several evolutionarily old plants (Salin and Bridges, 1981). Senescence was found to induce more intense bands of MnSOD. The induction of MnSODs, extracellular and mitochondrial, previously found in senescent pea (Jimenez et al., 1998), ginkgo, and birch leaves (Kukavica and Veljovic-Jovanovic, 2004), has been proposed to be part of a protective mechanism that accompanies an intensified respiration process. Combined activities of these two enzymes, the H2O2-generating SOD and H2O2-metabolizing POD, contribute to increasing drought resistance and delaying the senescence process. The present data on the contents of soluble proteins in Ramonda leaves showed that senescence and drought both induced a similar protein decrease expressed on a dry weight basis (Table 1). Senescence in perennial plants is accompanied by protein degradation as a consequence of intensified catabolic oxidative reactions and activation of specific proteases (Thomson et al., 1987). Contrasting results were reported on the protein levels in dehydrated leaves of resurrection plants, these being five to six times as high as in control leaves (Sgherri et al., 1994a), or reduced to 30% of the control value (Sgherri et al., 1994b). In dehydrated leaves, agreeing with some previous findings on other resurrection plants (Sgherri et al., 1994a), the SDS-PAGE analysis of soluble proteins in Ramonda leaves at 50% and 20% RWC showed two new polypeptide bands of around 25 and 35 kDa, while bands with 60 kDa and two below 15 kDa were intensified. Decrease in the amount of peptides in senescent leaves (Fig. 3) was probably due to a reported increase in amino acid contents in senescent leaves (Soudry et al., 2005). The existence of a close link between oxidative modification of proteins and their proteolysis has been suggested (Davies et al., 1987). On the other hand, the amino acid decrease in Ramonda leaves (
ivkovi
et al., 2004) may implicate a de novo protein synthesis (Fig. 3). Conservation of enzymes in desiccated leaves is a unique process unrelated to ageing, as obtained in senescent leaves, and it requires further study. Group 1 late embryogenesis-abundant proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during desiccation (Bartels and Salamini, 2001).
In conclusion, the present results confirm that dehydration, like senescence, causes disturbance in the redox homeostasis of the resurrection plant Ramonda by a preferential induction of class III peroxidase. Oscillatory changes in peroxidase activity (class III), accompanied by transient SOD and APX inactivation obtained during rehydration, confirm that plants experience a severe oxidative stress in the first few hours of restoration of the original membrane structure within cells (Navari-Izzo and Rascio, 1999; Quartacci et al., 2002). The isoperoxidase and peptide profile of dehydrated leaves and senescent leaves, compared with mature turgid leaves, supports an opinion that senescence and dehydration develop different mechanisms of protein protection when metabolism is slowed down. It is proposed that different POD isoforms induced under dehydration or senescence could have different physiological roles, anionic in lignifying cell wall, induced in senescence, and cationic in protecting cellular constituents in drought, oxidizing phenolics which are abundant in Ramonda leaves. The higher affinity of POD for hydroxycinnamic acids, compared with simple phenols (Fig. 4), and the unusually high level of chlorogenic and p-dihydroxybenzoic acids measured in Ramonda leaves (Sgherri et al., 2004) imply that such a peroxidase reaction occurs in situ. Decreases in phenolic acids and reduced ascorbate in dehydrated leaves (Sgherri et al., 2004), accompanied by an increase in soluble cationic isoperoxidase activity (Fig. 6), suggest that the enzymatic oxidation of hydroxycinnamic acids coupled to ascorbate oxidation by phenolic radicals (Takahama and Oniki, 1997) provides an efficient system for scavenging H2O2. In the absence of ascorbate in desiccated leaves, when transport from cytosol to apoplast and vacuole is impaired due to water loss and membrane disruption (Quartacci et al., 2002), o-quinones, oxidation products of chlorogenic and p-dihydroxybenzoic acids, can be either substrates for peroxidase in the presence of excess amounts of H2O2 (Zapata et al., 1992) or can have antimicrobial activity in the apoplast (Beckman and Siedow, 1985). Some of the oxidation products, o-diphenols can inhibit proteolysis, thus helping conservation of proteins in the anabiotic state of Ramonda leaves (Sullivan and Hatfield, 2006).
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Acknowledgements |
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This study was performed in collaboration with the University of Pisa (promoter F Navari-Izzo) and the University of Belgrade (promoter B Stevanovic). SV-J and BK acknowledge financial support of Ministry of Sciences, Technologies and Development of Serbia (Project No. 143020B).
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