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. 2003 Sep;92(3):385–391. doi: 10.1093/aob/mcg152

Photo- and Antioxidative Protection During Summer Leaf Senescence in Pistacia lentiscus L. Grown under Mediterranean Field Conditions

S MUNNÉ-BOSCH 1,*, J PEÑUELAS 1
PMCID: PMC4257514  PMID: 12871848

Abstract

Summer leaf senescence in Pistacia lentiscus L. plants serves to remobilize nutrients from the oldest leaves to the youngest ones, and therefore contributes to plant survival during the adverse climatic conditions typical of Mediterranean summers, i.e. water deficit superimposed on high solar radiation and high temperatures. To evaluate the extent of photo- and antioxidative protection during leaf senescence of this species, changes in carotenoids, including xanthophyll cycle pigments, and in the levels of ascorbate and α-tocopherol were measured prior to and during summer leaf senescence in 3-year-old plants grown under Mediterranean field conditions. Although a chlorophyll loss of approx. 20 % was observed during the first stages of leaf senescence, no damage to the photosynthetic apparatus occurred as indicated by constant maximum efficiencies of photosystem II photochemistry. During this period the de-epoxidation state of the xanthophyll cycle, and lutein, neoxanthin and ascorbate levels were kept constant. At the same time β-carotene and α-tocopherol levels increased by approx. 9 and 70 %, respectively, presumably conferring photo- and antioxidative protection to the photosynthetic apparatus. By contrast, during the later stages of leaf senescence, characterized by severe chlorophyll loss, carotenoids were moderately degraded (neoxanthin by approx. 20 %, and both lutein and β-carotene by approx. 35 %), ascorbate decreased by approx. 80 % and α-tocopherol was not detected in senescing leaves. This study demonstrates that mechanisms of photo- and antioxidative protection may play a major role in maintaining chloroplast function during the first stages of leaf senescence, while antioxidant defences are lost during the latest stages of senescence.

Key words: antioxidant, ascorbate, carotenoid, chlorophyll, leaf senescence, lentisc (Pistacia lentiscus L.), oxidative stress, photoprotection, tocopherol, xanthophyll cycle

INTRODUCTION

Senescence is an important developmental process in plants that leads to whole plant, organ, tissue or cell death through highly regulated, genetically controlled processes (Quirino et al., 2000; Chandlee, 2001). Leaf senescence is a key developmental state in the life of plants, as it is the time during which material built up by the plant during its growth phase is mobilized into younger tissues (e.g. growing leaves, flowers, fruits, developing seeds) to prepare for the next generation and/or to allow plant survival under adverse environmental conditions (Gan and Amasino, 1995; Buchanan et al., 2000). Leaf senescence is characterized by specific cell ultrastructural changes (Inada et al., 1998), and by chlorophyll loss (leaf yellowing), oxidative stress and decreases in photosynthesis (Dhindsa et al., 1981; Thompson et al., 1987; Leshem, 1988; Halliwell and Guterridge, 1989). Hence, chloroplasts are one of the first organelles to be targeted for breakdown as senescence proceeds, while nuclei and mitochondria maintain their integrity until the latest stages of senescence (Smart, 1994; Buchanan et al., 2000).

In plant cells, chloroplasts are one of the most important intracellular generators of activated oxygen species (AOS) such as singlet oxygen (1O2), superoxide radicals, hydrogen peroxide and hydroxyl radicals. AOS, especially 1O2 and hydroxyl radicals are highly reactive and, in the absence of protective mechanisms, can produce damage to cell structure and function (Elstner, 1991; Asada, 1999). Apart from the xanthophyll cycle, photorespiration and other changes in metabolic activity, which may protect the chloroplasts from oxidative damage (Demmig-Adams and Adams, 1992; Kozaki and Takeba, 1996; Eskling et al., 1997; Osmond et al., 1997), a number of enzymatic and non-enzymatic antioxidants are present in chloroplasts to control oxygen toxicity (Smirnoff, 1993; Foyer et al., 1994; Asada, 1999). Among the latter, carotenoids, α-tocopherol (α-T, vitamin E) and ascorbate (Asc, vitamin C) play an important role in maintaining the integrity of the photosynthetic membranes under oxidative stress (Smirnoff, 1996; Havaux, 1998; Munné-Bosch and Alegre, 2002a).

Lentisc (Pistacia lentiscus L., Anacardiaceae) is a dioecious sclerophyllous evergreen species that forms shrubs up to 2 m high, sometimes attaining a tree growth form in the more humid and protected sites, and is widely distributed along the Mediterranean basin. The longevity of leaves of P. lentiscus grown in a Mediterranean climate has been estimated to be somewhat longer than 1 year (Diamantoglou and Kull, 1988). Leaf senescence, which is followed by abscission, usually occurs during the summer in P. lentiscus grown in Mediterranean field conditions. Leaf senescence serves to remobilize nutrients from the oldest leaves to the youngest ones in P. lentiscus (Diamantoglou and Kull, 1988), which may contribute to plant survival during the adverse climatic conditions typical of Mediterranean summers, i.e. water deficit combined with high solar radiation and high temperatures (Pereira and Chaves, 1993).

The aim of this study was to evaluate the extent of photo- and antioxidative protection during summer leaf senescence in P. lentiscus plants. Changes in xanthophyll cycle pigments, and in the levels of ascorbate (a hydrophilic antioxidant) and α-tocopherol (a lipophilic antioxidant) were measured prior and during summer leaf senescence in 3-year-old P. lentiscus plants grown under Mediterranean field conditions. The results show that mechanisms of photo- and antioxidative protection may play a major role in maintaining chloroplast function during the first stages of leaf senescence, while antioxidant defences are lost during the latest stages of senescence, thus providing further evidence on the relationship between oxidative stress and leaf senescence.

MATERIALS AND METHODS

Plant material and growth conditions

Nine individuals of lentisc (Pistacia lentiscus L.), which were purchased in Garden Bordas (Gavà, Barcelona, Spain), were grown in pots of 1–5 l capacity (depending on root biomass as plants grew) containing a mixture of soil : peat : perlite (1 : 1 : 1, v/v/v). The pots were maintained in a glasshouse with controlled temperature (24/18 °C, day/night) and adequate watering conditions. After growing for 3 years, plants were transplanted to the experimental fields at the Universitat Autònoma de Barcelona (Bellaterra, Barcelona, north-east Spain) on 23 Feb. 2002. Plants were distributed evenly 1 m apart in an experimental flat 10 × 2 m2 plot, so that all plants had the same orientation to the sun. The experimental plot and its surroundings were always maintained clear of vegetation that could interfere in the growth of P. lentiscus. Before the experiment started (20 Mar. 2002), all plants were watered twice a week, so that they received approx. 100 mm month–1 (watering + rainfall). During the experiment, plants were grown under Mediterranean field conditions and received water from rainfall only.

The environmental conditions were monitored with a Davis weather station (Darrera S.A., Esplugues Ll., Barcelona, Spain) that was situated approx. 50 m from the experimental plot. Vapour pressure deficit (VPD) was calculated from air temperature (Ta) and relative humidity data according to Nobel (1991).

Sampling

Leaf samples from nine individuals were collected on one clear sunny day per month from March to July 2002. Water status, leaf mass area, the extent of lipid peroxidation [malondialdehyde (MDA) contents], chlorophyll fluorescence, photosynthetic pigments, α-T and reduced and oxidized Asc were measured in leaves collected at midday (at maximum incident diurnal PPFD). For measurements of MDA, photosynthetic pigments, α-T and reduced and oxidized Asc, leaves were collected, frozen in liquid nitrogen and stored at –20 °C until analysis.

Water status and leaf mass area

Leaves were weighed and leaf area was immediately measured using a flatbed scanner (model GT-5000; Epson, Nagano, Japan) and an image-processing program. Then, leaves were re-hydrated for 24 h at 4 °C in darkness and subsequently oven-dried for 24 h at 80 °C. The relative leaf water content (RWC) was determined as 100 × (FW – DW)/(TW – DW), where FW is the fresh weight, TW is the turgid weight after re-hydrating the leaves, and DW is the dry weight after oven-drying the leaves. The leaf mass area was determined as DW/leaf area.

Estimation of lipid peroxidation

The extent of lipid peroxidation in leaves was estimated by measuring the amount of MDA by the method described by Hodges et al. (1999), which takes into account the possible influence of interfering compounds in the assay for thiobarbituric acid (TBA)-reactive substances. In short, samples were repeatedly extracted with 80 : 20 (v/v) ethanol/water containing 1 ppm butylated hydroxytoluene (BHT) using sonication for 45 min at 4 °C (ultrasonic bath Type T570/H; Elma, Singen, Germany). After centrifugation, supernatants were pooled and an aliquot of appropriately diluted sample was added to a test tube with an equal volume of either (a) –TBA solution containing 20 % (w/v) trichloroacetic acid and 0·01 % (w/v) BHT, or (b) +TBA solution containing the above plus 0·65 % (w/v) TBA. Samples were heated at 95 °C for 25 min and, after cooling, absorbance was read at 440 nm, 532 nm and 600 nm. MDA equivalents (nmol ml–1) were calculated as 106 × [(A – B)/157000], where A = [(Abs 532+TBA) – (Abs 600+TBA) – (Abs 532–TBA – Abs 600–TBA)], and B = [(Abs 440+TBA – Abs 600+TBA) × 0·0571].

Chlorophyll fluorescence

Measurements of the maximum efficiency of PSII photochemistry (Fv/Fm) were conducted in situ on attached leaves with a portable pulse-modulated fluorimeter PAM-2000 (Walz, Effeltrich, Germany). Leaves were dark-adapted with leaf clips for 1 h, which was determined to be sufficient to allow complete relaxation of energy-dependent quenching. The Fv : Fm ratio was calculated as (Fm – Fo)/Fm, where Fm and Fo are the maximum and basal fluorescence yields, respectively, of dark-adapted leaves (Genty et al., 1989).

Photosynthetic pigments and α-tocopherol

The extraction and HPLC analyses of photosynthetic pigments and α-tocopherol was carried out essentially as described in Munné-Bosch and Alegre (2000). In short, leaves were repeatedly extracted with ice-cold 85 % (v/v) acetone and 100 % acetone using sonication for 45 min at 4 °C (ultrasonic bath Typ T570/H; Elma). Pigments were separated on a Dupont non-endcapped Zorbax ODS-5 µm column (250 × 4·6 mm, 20 % C; Scharlau, Barcelona, Spain) at 30 °C at a flow rate of 1 ml min–1. The solvents consisted of (A) acetonitrile/methanol (85 : 15, v/v) and (B) methanol/ethyl acetate (68 : 32, v/v). The gradient used was: 0–14 min 100 % A, 14–16 min decreasing to 0 % A, 16–28 min 0 % A, 28–30 min increasing to 100 % A, and 30–38 min 100 % A. Detection was carried out at 445 nm (diode array detector 1000S; Applied Biosystems, Foster City, CA, USA). α-T was separated on a Partisil 10 ODS-3 column (250 × 4·6 mm; Scharlau) at a flow rate of 1 ml min–1. The solvents consisted of (A) methanol/water (95 : 5, v/v) and (B) methanol. The gradient used was: 0–10 min 100 % A, 10–15 min decreasing to 0 % A, 15–20 min 0 % A, 20–23 min increasing to 100 % A, and 23–28 min 100 % A. α-T was quantified through its absorbance at 283 nm (diode array detector 1000S; Applied Biosystems). Compounds were identified by their characteristic spectra and by coelution with authentic standards, which were obtained from Fluka (Buchs, Switzerland).

Reduced and oxidized ascorbate

The extraction and HPLC analysis of reduced and oxidized Asc was carried out as described in Munné-Bosch and Alegre (2002b). In short, leaves were repeatedly extracted with ice-cold extraction buffer [40 % (v/v) methanol, 0·75 % (w/v) m-phosphoric acid, 16·7 mm oxalic acid, 0·127 mm diethylenetriaminepentaacetic acid] by using sonication for 45 min at 4 °C (ultrasonic bath Typ T570/H; Elma). The extract was centrifuged for 10 min at 3 °C and 6000 g, and 0·1 ml of the supernatant was transferred to 0·9 ml of the mobile phase [24·25 Na–acetate/acetic acid, pH 4·8; 0·1 mm diethylenetriaminepentaacetic acid; 0·015 % (w/v) m-phosphoric acid; 0·04 % (w/v) octylamine; 15 % (v/v) methanol]. For determination of total Asc (reduced plus oxidized; Asct) 0·1 ml of the supernatant was incubated for 10 min at room temperature in darkness with 0·25 ml of 2 % (w/v) dithiothreitol and 0·5 ml of 200 mm NaHCO3. The reaction was stopped by adding 0·25 ml of 2 % (v/v) sulfuric acid and 0·8 ml of the mobile phase. Asc was isocratically separated on a Spherisorb C8 column (250 × 4·6 mm; Teknokroma, St Cugat, Spain) at a flow rate of 0·8 ml min–1. Detection was carried out at 255 nm (diode array detector 1000S; Applied Biosystems). Asc was identified by its characteristic spectrum and by coelution with an authentic standard from Sigma (Steinheim, Germany).

Statistical analyses

Statistical differences between measurements on different days were analysed following one-way ANOVA using SPSS (Chicago, IL, USA). Differences were considered significant at a probability level of P < 0·05.

RESULTS

Summer leaf senescence in field-grown P. lentiscus plants

Environmental conditions during the experimental period (March–July 2002) were typical of a Mediterranean climate (Fig. 1). From the beginning of March to the beginning of June, precipitation was abundant (252 mm). However, from mid-June to the end of July, precipitation was scarce (7·6 mm), and this was accompanied by increases in the maximum diurnal photosynthetically active photon flux density (PPFD) up to approx. 2100 µmol m–2 s–1, midday air temperatures (Ta) above 30 °C, and pressure deficit (VPD) values ranging between 2 and 4 KPa at midday. Despite the low precipitation during July, the relative leaf water content (RWC) was maintained above 80 %. The RWC changed only slightly throughout the study period, and the small variations observed negatively correlated with the VPD (slope = –2·61, r2 = 0·863).

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Fig. 1. Environmental conditions and relative leaf water content (RWC) of Pistacia lentiscus plants from March to July 2002. Vapour pressure deficit, photosynthetically active photon flux density (PPFD), air temperature (Ta) and RWC correspond to measurements made at midday (at maximum diurnal PPFD). RWC data correspond to the mean ± s.e. of nine individuals, and five bulked leaves per plant were measured. Rainfall during the experimental period is shown in bars.

The first symptoms of leaf senescence were observed between April and May, during which period leaf biomass, chlorophyll a+b (Chl) contents and the Chl a/b ratio decreased significantly to a similar extent (between 20 and 25 %) (Fig. 2). Leaf area also decreased significantly during this period, but to a lesser extent than leaf biomass, which caused decreases in the leaf mass area. Despite these first symptoms of senescence, MDA levels and the Fv/Fm ratio were kept constant between March and May. Decreases in leaf biomass, Chl contents and the Chl a/b ratio were especially severe between May and June, with minimum values attained during June and July. During this period, leaf biomass, Chl and the Chl a/b ratio were between 60 and 68 % lower than in March. Besides, significant increases in MDA levels (by approx. 33 %) and significant decreases in the Fv/Fm ratio (by approx. 15 %) were observed between May and June (Fig. 2). During July and August, leaf abscission was observed in the field. Leaf abscission during this period varied among individuals in the range between 20 and 30 % of the total leaf biomass (data not shown).

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Fig. 2. Changes in leaf biomass, leaf mass area (LMA), chlorophyll a+b (Chl) contents, Chl a/b ratio, malondialdehyde (MDA) levels and the maximum efficiency of photosystem II photochemistry (Fv/Fm) in leaves of Pistacia lentiscus plants grown under Mediterranean field conditions from March to July 2002. Data correspond to the mean ± s.e. of nine individuals, and five bulked leaves per plant were measured.

Photo- and antioxidative protection during leaf senescence

The amount of xanthophyll cycle pigments, i.e. violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z), in leaves decreased progressively from April to July (Fig. 3). The amount of total xanthophyll cycle pigments (V + A + Z) decreased up to approx. 60 % in senescing leaves. V was the xanthophyll showing the highest decreases. While both Z and A decreased up to approx. 55 %, V was almost completely degraded (it decreased up to approx. 99 %), in senescing leaves. The de-epoxidation state of the xanthophyll cycle [DPS, given as (Z + 0·5A)/(V+Z+A)], which was high at the beginning of the experiment, increased by approx. 10 % during the latest stages of leaf senescence (Fig. 3).

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Fig. 3. Changes in xanthophyll cycle pigments and the de-epoxidation state of the xanthophyll cycle (DPS) in leaves of Pistacia lentiscus plants grown under Mediterranean field conditions from March to July 2002. Data correspond to the mean ± s.e. of nine individuals, and five bulked leaves per plant were measured. V, violaxanthin; VZA, violaxanthin + zeaxanthin + antheraxanthin. DPS, calculated as (Z + 0·5A)/VZA.

Changes in the levels of low-molecular-weight antioxidants (carotenoids, α-T and Asc) showed that, while during the first symptoms of senescence (May) the leaves maintained or even enhanced their antioxidative protection, they showed a severe and significant loss of antioxidant defences during the latest stages of senescence (Fig. 4). While lutein, neoxanthin and Asc levels were kept constant until May, β-C and α-T levels increased by approx. 9 % and 70 %, respectively, from April to May, coincident with the first symptoms of senescence. During the latest stages of leaf senescence, both these low-molecular-weight antioxidants decreased, though to a different extent. While carotenoids were moderately degraded (neoxanthin by approx. 20 %, and both lutein and β-C by approx. 35 %), Asc decreased by approx. 80 % and α-T was not detected in senescing leaves (June and July). Despite the strong Asc decreases, oxidized Asc was not detected throughout the study period in P. lentiscus leaves (data not shown). Inter-individual differences in leaf biomass, the concentrations of Chl, xanthophyll cycle pigments, α-T and Asc, and the Fv/Fm ratio were also analysed. Individuals of P. lentiscus did not show consistent differences in the parameters analysed throughout the study period (data not shown).

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Fig. 4. Changes in the levels of lutein (L), neoxanthin (N), β-carotene (β-C), α-tocopherol (α-T) and reduced ascorbate (Asc) in leaves of Pistacia lentiscus plants grown under Mediterranean field conditions from March to July 2002. Data correspond to the mean ± s.e. of nine individuals, and five bulked leaves per plant were measured.

DISCUSSION

Summer leaf senescence in P. lentiscus plants grown under Mediterranean field conditions was characterized by Chl loss (leaf yellowing), decreases in the Chl a/b ratio and a loss of leaf biomass. These senescence symptoms were first described by Diamantoglou and Kull (1988) for the same species growing under similar environmental conditions in Greece. The same authors showed that leaf senescence in this species serves to remobilize nutrients (especially nitrogen) from the oldest leaves to the youngest ones. In the present study, it is shown that mechanisms of photo- and antioxidative protection may play a major role in maintaining chloroplast function (i.e. nutrient remobilization) during the first stages of leaf senescence, while antioxidant defences are lost during the latest stages of senescence in leaves of P. lentiscus.

Summer leaf senescence favours plant survival in several Mediterranean species. It serves to remobilize nutrients (Diamantoglou and Kull, 1988) and, when accompanied by leaf abscission, it helps to prevent plant desiccation (Kozlowski, 1976; Proebsting and Middleton, 1980). While in some species (e.g. in Salvia officinalis), leaf senescence is accelerated by a decrease in the leaf water content caused by summer droughts (Munné-Bosch et al., 2001), in others (e.g. in P. lentiscus) leaf senescence is not accompanied by a loss of leaf turgor (Diamantoglou and Kull, 1988). In the present study, leaf water content in P. lenstiscus was also maintained constant during senescence. Despite low precipitation occurring from mid-June to the end of July, the RWC was kept above 80 % at midday, and changed only slightly throughout the study period, in parallel with the VPD. Summer leaf senescence in P. lentiscus could be favoured by high solar radiation rather than by a loss of leaf turgor. The first symptoms of senescence were observed during May, when PPFD at midday was above 1800 µmol m–2 s–1. The combination of high solar radiation with high temperatures at midday (above 30 °C during several days of June and July) might also contribute to the physiological changes observed during the latest stages of leaf senescence.

The decreases in Chl levels and in the Chl a/b ratio between April and May were the first symptoms of senescence observed in chloroplasts of P. lentiscus leaves. A decline in the Chl a/b ratio suggests preferential loss of Chl a-containing proteins closely associated with the reactions centres rather than loss of light harvesting proteins (Lichtenthaler, 1987). This has also been observed during autumn foliar senescence in Larix occidentalis (Rosenthal and Camm, 1997) and during drought-induced senescence in Salvia officinalis (Munné-Bosch et al., 2001). Despite Chl loss and decreases in the Chl a/b ratio, MDA levels and the Fv/Fm ratio were kept constant between April and May. During this period, lutein, neoxanthin and Asc levels were kept constant, while levels of β-C and α-T increased, which might contribute to maintainance of the structure and function of photosynthetic membranes during the first stages of leaf senescence. It has been shown that α-T levels increase during Mediterranean summers in drought-resistant shrubs (Munné-Bosch and Alegre, 2000, 2002c). The cooperation between α-T and β-C seems to be essential to scavenge efficiently singlet oxygen produced in the reaction centre of photosystem II, which would otherwise trigger the degradation of the D1 protein (Trebst et al., 2002). Besides, α-T, in combination with Asc (which is needed for α-T recycling), scavenges lipid peroxyl radicals, thus avoiding propagation of lipid peroxidation in photosynthetic membranes (Munné-Bosch and Alegre, 2002a).

A severe loss of antioxidant defences, especially of α-T and Asc, but also of carotenoids, was observed during the latest stages of leaf senescence (June and July) in P. lentiscus. During this period, depletion of antioxidants was associated with increased MDA levels and decreases in the Fv : Fm ratio, which indicates enhanced oxidative stress and photoinhibitory damage to the photosynthetic apparatus. α-T deficiency might trigger the degradation of the D1 protein by singlet oxygen and, therefore, the disassembly of photosystem II subunits. α-T deficiency might also favour propagation of lipid peroxidation, thus favouring disassembly of photosynthetic membranes in senescing leaves. Asc deficiency correlated with severe α-T and carotenoid losses in senescing leaves, which is indicative of the interplay between lipophilic and hydrophilic low-molecular-weight antioxidants in plants (Smirnoff, 1996; Asada, 1999). β-C, which is very efficient in the scavenging of triplet chlorophyll in antenna complexes and in the quenching of singlet oxygen (Cogdell and Frank, 1988), was also significantly depleted, which might also favour disassembly of chloroplasts during the latest stages of senescence.

During the first stages of leaf senescence (between April and May), the levels of xanthophyll cycle pigments decreased, but the DPS was kept constant, since V decreased to a greater extent than A and Z. The xanthophylls, and especially Z have been ascribed a protective function, light- and drought-adapted plants displaying a larger pool of xanthophylls and a greater maximal conversion to Z (Thayer and Björkman, 1990; Demmig-Adams and Adams, 1992; Logan et al., 1996; Munné-Bosch and Alegre, 2000). However, only a few molecules of Z per reaction centre of photosystem II, in combination with a pH gradient across the thylakoid membrane are required for photoprotective energy dissipation (Bukhov et al., 2001; Heber et al., 2001). Besides, the DPS, rather than Z accumulation, has been correlated with thermal dissipation of excess excitation energy (Demmig-Adams and Adams, 1992), which suggests that dissipation of excess excitation energy as heat by the xanthophyll cycle remained constant during the first stages of leaf senescence, and increased slightly during the latest stages of senescence in leaves of P. lentiscus. Besides, decreases in xanthophyll cycle pigments may play a role in senescing leaves. Z has been suggested to protect thylakoid membrane lipids from AOS under stress (Havaux et al., 1991), thus the severe loss of Z may also contribute to the oxidative stress observed during the latest stages of leaf senescence. Besides, V and neoxanthin decreases might be associated with abscisic acid synthesis (Qin and Zeevaart, 1999), which is known to accelerate leaf senescence (Chen et al., 2002; Yang et al., 2002). Finally, Z is thought to participate in the blue light response (Quiñones and Zeiger, 1994), and influence thylakoid membrane fluidity (Gruszecki and Strzalka, 1991), which might also affect senescence in P. lentiscus leaves.

Despite the large depletion of Asc concentrations during the latest stages of leaf senescence, dehydroascorbate was not detected throughout the study period in P. lentiscus. To our knowledge this is the first report on Asc concentrations in leaves of this species, and further research is needed to elucidate the causes, by which (a) this species does not accumulate dehydroascorbate, or (b) this compound cannot be detected in leaves of this species.

In conclusion, the results show that mechanisms of photo- and antioxidative protection play a major role in maintaining chloroplast function (i.e. nutrient remobilization) during the first stages of leaf senescence, while a severe loss of antioxidant defences and, therefore, oxidative stress occur during the latest stages of senescence in P. lentiscus leaves.

ACKNOWLEDGEMENTS

We are very grateful to the Serveis Científico-Tècnics (University of Barcelona) for technical assistance. This research was supported by MCYT-REN2000-0278/CLI and MCYT-REN2001-0003/GLO grants from the Spanish Government and by the European Environment Programme (VULCAN-EVK2-CT-2000-00094 grant).

Received: 14 February 2003; Returned for revision: 14 May 2003; Accepted: 29 May 2003Published electronically: 18 July 2003

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