OXIDATIVE STRESS AND ASCORBIC ACID CONTENTS INPARMOTREMA RETICULATUMANDPARMELIA SULCATATHALLI

Lichenologist 31(1): 105-110 (1999) Article No. lich. 1998.0164 Available online at http://www.idealibrary.com on

OXIDATIVE STRESS AND ASCORBIC ACID CONTENTS IN PARMOTREMA RETICULATUM AND PARMELIA SULCATA THALLI A. M. CAVIGLIA* and P. MODENESI* Abstract: Paraquat-treatment of Parmotrema reticulatum and high light levels in the habitat of Pamtelia sulcata can cause oxidative stress, which promotes an increase in ascorbic acid contents of thalli. Ascorbic acid was measured by an HPLC method. In P. reticulatum the increase was more pronounced in summer than in other seasons. In P. sulcata, thalli growing in sun-exposed habitats showed a higher ascorbic acid content than shade-growing specimens. '£ 1999 The British Lichen Society

Introduction It has been suggested that the production of heavy crystalline deposits of calcium oxalate at the surface of epiphytic lichens could be linked to exposure to oxidative stress (Modenesi 1993; Modenesi et al. 1998). The observations were carried out on SO2-stressed and paraquat-treated thalli of Parmotrema reticulatum (Modenesi 1993) and on thalli of Parmelia sulcata growing in a sun-exposed habitat. The latter thalli showed a heavy oxalate pruinosity above the cortex, in comparison with thalli growing in a shade habitat that lacked pruina (Modenesi et al. 1998). SO2-pollution, paraquat-treatment and highlight-intensity environments, all promote pruina deposition, a response to toxicity involving the generation of active oxygen species. In green plants, paraquat is reduced rapidly by Photosystem I in the light to give paraquat radical ions P' + (the • denotes an unpaired valency electron). These radicals rapidly auto-oxidize by the transfer of single electrons to oxygen, thus producing superoxide O' ~~ (see for paraquat toxicity Babbs et al. 1989; for SO2 Peiser & Yang 1977; Richardson & Nieboer 1983; Takahama et al. 1992; for photo-oxidative stress Long & Humphries 1994; Tschiersch & Ohmann 1993; for oxygen toxicity Elstner 1982; Halliwell & Gutteridge 1989). The lichen thallus may not ' distinguish' between the different situations reported above but, recognizing the common oxidative stress as equivalent to high irradiance, it responds by depositing whitish layers of calcium oxalate, which increases the surface albedo of the thallus (Modenesi et al. 1998). To explain the possible link between oxidative stress and oxalate deposition in lichens we have hypothesized (Modenesi 1993) that the primary event in the thallus could be an increased synthesis of ascorbic acid, a compound well known as an antioxidant (Keller & Schwager 1977) and, with glyoxylate, the major precursor of oxalic acid in plants and fungi (Loewus 1988). *Istituto Botanico ' Hanbury ', University of Genoa, Corso Dogali 1/c, 1-16136 Genova, Italy. 0024-2829/99/010105 + 06 $30.00/0

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The present study compares the ascorbic acid content, measured by an HPLC method, in thalli of P. reticulatum and P. sulcata subjected to oxidative stress. Materials and Methods Sample collection and treatment Parmotrema reticulatum (Taylor) M. Choisy was collected on S. Ambrogio hill, maximum elevation 170 m, near Rapallo in East Liguria (north-west Italy), on the Ligurian coast. Thalli growing on olive trees were collected in a clean area zone, as described elsewhere (Modenesi 1993). Some thalli were collected before treatment and used as controls (untreated thalli), others were treated with paraquat (Modenesi 1993). Each thallus was treated as follows: an area surrounding the sample was sprayed with 50 ml dm ~ 2 of 0-2% paraquat dissolved in deionized water. Technical grade paraquat (Gramoxone W, ICI Solplant) was used as recommended by the manufacturer (3-5 kg ha " '). Lichen thalli were sprayed only once and collected 1, 2 and 4 weeks after treatment. Seven thalli for each ascorbic acid determination were used; a total of 84 thalli were used for the experiments. The experiments were performed in duplicate, in April, July and October to evaluate the seasonal variations in ascorbic acid content. Parmelia sulcata Taylor was collected in the Visone river valley in Lower Piedmont (north-west Italy) in two sites 200 m away from each other, referred to as shade and sun habitats. Ten thalli, collected in summer from each habitat, were used for each ascorbic acid determination. The shade habitat site faced north-west, 200 m a.s.l., in a Quercus pubescens deciduous wood. The thalli were collected on oak bark. The sun habitat site was in an open field at the top of a hill facing south-east, 350 m a.s.l. The thalli were growing on branches of old, scattered bushes of Crataegus monogyna (see for details Modenesi et al. 1998). Analytical methods The method employed was originally described by Wimalasiri & Wills (1983) for fresh fruit and vegetables, but was modified to quantify accurately the ascorbic acid on a microscale in lichen material. All samples of P. reticulatum and P. sulcata were thoroughly cleaned of tree bark fragments and other debris in the laboratory immediately after collection. Cleaned samples were dried for 24 h above silica gel in a desiccator, to standardize water contents, in the dark at room temperature. Dry material was divided into 100-mg samples and stored at - 20°C until use. Lichen material was also oven-dried at 60°C for 48 h and weighed to obtain the percentage dry weight. Extraction of ascorbic acid Samples of frozen thalli were rapidly homogenized in 4-ml ice-cold 2-5 mM o-phosphoric acid at 3000 x g, for 2 min in the dark. The stability of ascorbic acid in o-phosphoric acid solution (after 1 year at — 20°C recovery was 98-7%) and the lack of interference due to the extracting acid, were tested (unpublished data). The homogenate was shaken for 2 min and centrifuged for 5 min at 3000 x g. The resulting supernatant was filtered through Whatman no. 1filterpaper and purified by passage through Sep-Pak C18 cartridges (Waters, USA), preconditioned by washing with methanol (4 ml) and water (10 ml). Aliquots (0-5 ml) of the purified extract were used directly to determine ascorbic acid content. HPLC determination A Perkin-Elmer (Norwalk, USA) HPLC equipment with UV detector and a Shimadzu (Kyoto, Japan) data processor were used. Sample injection system: Rheodyne valve with 20-ul sample loop. Column: Nucleosil 10 C18 (Macherey-Nagel, Germany). Mobile phase: Acetonitrile-water (35-65) acidified with 0-1 M o-phosphoric acid (25 ml 1 ') and containing 2 mM tetrabutylammonium hydroxide (Kodak, USA) as ion-pair reagent (final pH = 4-3). Flow rate: 0-7 ml min ~ '. Detector: UV 254 nm. Range: 0-004 AUFS. Ascorbic acid was determined by reference to authentic L (+)-ascorbic acid (Aldrich, England). The external standard method was used.

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TABLE 1. Percentage dry weight (dry wt. %) and ascorbic acid (AA) content of thalli of Parmotrema reticulatum collected in different seasons, before and after different times following a single paraquat treatment Time from treatment 0 week 1 week 2 weeks 4 weeks

April

October

July

Dry wt. %

AA*

Dry wt. %

93-0 90-9 94-1 94-8

4 •76 ± 0 • 9 6 7 •65 ± 0 • 7 8 3 •74 ± 0 • 6 0

93-3 92-5 95-7 95-4

—

AA 9 •40 ± 1 •47 21 •07 ± 0 •8 1•90 ± 0 • 7 8 —

Dry wt. %

AA

91-8 91-9 94-1 94-2

6-00 ± 0 •87 8-23 ± 1 •09 5-54 ± 0 •90 —

*mg 100 g ' dry wt. Data are mean ± standard error (n = 7). —=undetectable.

The detector response was linear in the range 2-2000 ng per 20 (il samples, and the chromatographic system was able to detect 0-5 ng of ascorbic acid in 20 ul injected. Recovery of the ascorbic acid standard solution was 5810% ± 1-09 (n = 5); lichen data were corrected for this value. The data obtained were statistically processed by using a standard analysis of variance (ANOVA) for determining significant intergroup differences (Barnard et al. 1993).

Results and Discussion Ascorbic acid contents in paraquat-treated and untreated (0 week in Table 1) thalli of P. reticulatum showed wide fluctuations seasonally depending on collection periods, as shown in Table 1. Untreated thalli collected in summer showed an increase of 97% or 56% in ascorbic acid content in comparison to those collected, respectively, in spring and autumn. After a week of paraquat treatment, all thalli showed an increase in ascorbic acid content of between 37% in autumn samples and 124% of summer ones. Spring samples had an intermediate increase of 60%. Two weeks after paraquat treatment all samples showed a strong decrease, which was greatest in those collected in summer. Ascorbic acid content 4 weeks after paraquat treatment was below the detection limit of the instrument. The two-way ANOVA showed that intergroup differences (in ascorbic acid contents) were significant (P