Introduction

• IO ••• ALOF. PI ....... , ••• .., © 1998 by Gustav Fischer Verlag, Jena

Reducing Properties, and Markers of Lipid Peroxidation in Normal and Hyperhydrating Shoots of Prunus avium L.

T. FRANCK1,2, c. KEVERSl, c. PENEL3, H. GREPPIN3, J. F. HAUSMAN2, and T. GASPARI

1 Hormonologie Fondamentale et Appliquee, Institut de Botanique B 22, Universite - San TIlman, B-4000 Liege, Belgium

2 CREBS, Centre de Recherche Public-Centre Universitaire, 162a, Avenue de la Falencerie, L-1511 Luxembourg 3 Laboratoire de Physiologie Vegetale, Universite de Geneve, Place de l'Universite 3, CH-1211 Geneve 4, Switzerland

Received August 25,1997 . Accepted November 3, 1997

Summary

The amounts of some reductants (ascorbic acid, reduced glutathione, a-tocopherol) and the amounts of some markers of lipid peroxidation (peroxide and malondialdehyde) were quantified weekly in normal shoots (NS, in culture on agar) and in hyperhydrating shoots (HS, in culture on gelrite) of Prunus avium L. The redox activity of the plasma membrane (reduction of exogenously added ferricyanide), the antilipo­peroxidant potential, the level of hydrogen peroxide and the lipoxygenase (EC 1.13.11.12) activity were investigated after 28 days of culture in both types of shoots. Reducing capacity of HS seemed generally more efficient in comparison to NS: higher levels of free ascorbate, reduced glutathione and the antilipo­peroxidant potential were measured in HS than in NS. The a-tocopherol content did not change between the two types of shoots Reduction of exogenously applied ferricyanide was lower in HS during the last 2 weeks of the culture. These results suggest that the plasma membrane of HS had an unchanged reducing capacity but less redox transfer activity in comparison to NS. Markers of membrane damage (peroxide and malondialdehyde) were lower in HS than in NS and the same level of hydrogen peroxide was measured in the two types of shoots. Therefore, HS seem not to be submitted to oxidative stress. However, a more important lipoxygenase activity measured in HS was in contradiction to the lower peroxidation of lipids. The discussion points out some paradoxical results in an extensive classical analysis of stress criteria and indicates alternative defense mechanisms.

Key words: Defense systems, gelrite, hydrogen peroxide, hyperhydricity, peroxitlation, redox capacity, Prunus avium L.

Abbreviations: AA = ascorbate; DHA = dehydroascorbate; GSH = reduced glutathione; GSSG = oxidiz­ed glutathione; H 20 2 = hydrogen peroxide; HS = hyperhydrating shoots; LOX = lipoxygenase; MDA = malondialdehyde; NS = normal shoots; NAD{P)H = reduced nicotinamide adenine dinucleotide phos­phate; PUPA = polyunsaturated fatty acid; TBA = thiobarbituric acid.

Hyperhydricity (previously known as vitrification) is a physiological disorder frequently affecting in vitro propagated shoots (Gaspar, 1991; Debergh et al., 1992). Leaves of hyper­hydric shoots are thick, frequently very elongated, wrinkled and/or curled, and brittle. Sterns are broad and thick in diameter, and internodes are shorter than those of plants appearing normal. The phenomenon of hyperhydricity was ofren considered as a physiological response due to simulta-

neous abnormal conditions: e.g. high amount of cytokinins, high ammonium content in the culture medium, high rela­tive humidity in the flask atmosphere, and/or accumulation of specific gases in the confined atmosphere (Kevers et al., 1984; Ziv, 1991; Gavidia et al., 1997). In most plants sub­jected to stress, a variety of toxic oxygen species (e.g. oxygen superoxide anion, hydroxyl radical, singlet oxygen) and/or H 20 2 are produced, which may lead to severe damage of cell molecules, membranes and other structures (&ada, 1992). These substances are generally eliminated through enhanced

J Plant PhysioL WlL 153. pp. 339-346 (1998)

340 T. FRANCK, C. KEvERS, C. PBNBL, H. GRBPPIN, J. F. HAUSMANN, and T. GASPAR

activities of defense enzymes such as SOD (EC 1.15.1.1), which converts the oxygen superoxide anion to H 20 2, per­oxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and the Halli­well-Asada pathway (Foyer and Halliwell, 1976; Asada and Takahashi, 1987), which scavenges H 20 2• The Halliwell­Asada pathway ensures the elimination of H20 2 within the chloroplasts by ascorbate peroxidase (EC 1.11.1.11) oxidizing ascorbate to ascorbate free radicals (MDHA). MDHA can be spontaneously reduced to AA and DHA, or can be enzymat­ically reduced by monodehydroascorbate reductase (EC 1.6.5.4) utilizing NAD{P)H as reductant. Ascorbate is then regenerated in a GSH dependent reaction catalysed by dehydroascorbate reductase (EC 1.8.5.1). The GSSG is then reduced back to GSH in a reaction involving glutathione reductase (EC 1.6.4.2) and NAD{P)H Qahnke et al., 1991; Polle et al., 1992). In hyperhydrating shoots (HS), all of the defense enzymes listed above, except SOD, had lower activ­ities than in normal shoots (NS) (Franck et al., 1995). Ab­normal morphology of HS of Prunus 'avium L. was recently characterized by reduced chlorophyll content, chloroplast de­generation by lytic phenomenon and membrane residues in the intercellular spaces (Franck et al., 1997). This supports the hypothesis that morphological abnormalities that charac­terize hyperhydricity result from an accumulation of toxic oxygen forms and/or H20 2 caused by the inability of in vitro shoots to adapt to hyperhydrating (stress) conditions by mo­bilizing a defense system (Sankhla et al., 1994; Franck et al., 1995). The aim of the present work was to test this hypothe­sis in HS and NS of Prunus avium L. by studying: 1) the reducing capacity (ascorbic acid, reduced glutathione,

a-tocopherol, and antilipoperoxidant level) against free toxic oxygen and radical forms,

2) the redox activity of the plasma membrane by reduction of exogenously added ferricyanide, and

3) an element involved in membrane damage (H20 2) and markers of lipid peroxidation (peroxide, malondialdehyde and lipoxygenase).

Materials and Methods

Hyperhydrating shoots (HS) of Prunus avium L. were obtained through one culture cycle of 4 weeks by a simple transfer of normal shoots (NS) on the same medium where agar (8~L -1) (Roland Brus­sels, Belgium) was replaced by gelrite (2.5 g L -) (Carl Roth com­pany, Karlsruhe, Germany). Symptoms of hyperhydricity (translu­cent stems and leaves; wrinkled, curled and thicker leaves) were ap­parent at day 7 on about 35 % of the shoots on culture with gelrite. On day 21 of the 28-day culture period, 100 % of the shoots were hyperhydric (Franck et al., 1995).

Danmination of ascorbate and dehydroascorbate

Three shoots (250 mg of fresh material) were homogenized in 2 mL of cold 5 % (w/v) trichloroacetic acid (TCA) containing 100 mg insoluble polyvinylpytrolidone (PVP) and 100 mg of quartz sand. The homogenate was filtered through 4 layers of Miracloth and centrifuged at 16,000 g for 10 min at 4 .c. The supernatant was used for AA and total ascorbate (AA + DHA) assay using the method of Wang et al. (1991). This assay is based on the reduction of ferric ion to ferrous ion with ascorbic acid followed by formation of the red chelate between ferrous ion and 4,7-diphenyl-l,1O-

phenanthroline (bathophenanthroline) that absorbs at 534 nm. Total ascorbate was determined through a reduction of D HA to AA by di­thiothreitol. DHA concentration was estimated from the difference of total ascorbate and AA concentrations.

Dtttrmination of rtductd and oxidized glutathiont

Fresh shoots (250mg) were ground in a mortar under N2 and ho­mogenized in 2 mL ice cold 8 mmoVL sodium ascorbate solution. The homogenate was centrifuged (30,000 g, 15 min, 4 0C) and the supernatant was deproteinized twice, according to Wang et al. (1991), in glass test tubes by incubation in a water bath at 100·C for 3 min then by centrifugation at 15,000gfor 15min at 4°C. The su­pernatant was used as extract. GSH was oxidized by 5, 5'-dithio­bis(2-nitrobenwic acid) (DTNB) to give GSSG with the formation of 2-nitro-5-thiobenzoic acid (TNB). GSSG was reduced to GSH by action of the highly specific glutathione reductase and NADPH. TNB formation was followed as the rate change in absorbance at 412 nm and was proportional to total glutathione (GSH + GSSG). Oxidized glutathione was determined after removal of reduced glu­tathione with N-ethylmaleimide (NEM). GSH was determined by the subtraction of GSSG from total glutathione.

Dtttrmination of a-tocophtrol

Fresh shoots (500 mg) were ground in a mortar with 1 mL of methanol and 3 mL of hexane for UV analysis. The mortar was rinsed with 2 mL of methanol. The homogenate was mixed for 1 min and centrifuged at 500 g for 10 min at 4 0c. The upper he­xane green phase was collected and filtered (1.5 mL) on a C18 col­umn rinsed with 1.5 mL of hexane. The extract was used for a-to­copherol determination and methanol was added up to 1.95 mL for fluorescence measurement (excitation wavelength: 292 nm, emission wavelength: 329 nm) according to Undenfriend (1962).

Ftrricyanide rtduction

Fresh shoots (250 mg) were incubated at 25°C according to Carrie et al. (1994) in 2 mL of 0.1 mmol/L 2-morpholino-ethane­sulphonic acid (MES) containing 1 mmoVL ferricyanide. The infil­tration of the incubation mixture in the apoplast was facilitated by several passages through vacuum (-80 K Pa). After a period of 90 min, a 1 mL aliquot was withdrawn. The absorbance of the ali­quot was determined spectrophotometrically at 412 nm and calcu­lated using a millimolar extinction coefficient of 1.00 according to Malerba et al. (1995).

Ma/ondialdehyde (MDA) contmt

Lyophilised shoots (500 mg fresh weight) were ground in 1 mL (10 % w/v) TCA. After several washings with acetone and centrifu­gations (4,000 g, 10 min), the resulting pellet was incubated at 100 ·C for 30 min with 3 mL H3P04 (1 %) and 1 mL ofTBA 0.6 % and then cooled in ice. Three mL n-butanol was added and the re­sulting mixture was agitated and centrifuged (4,000 g, 10 min). The persistence of the butanolic layer was evaluated by measuring the difference between the absorbance at 532 nm and 590 nm according to Hagege et al. (1990b).

PtroXide index

Lyophilised shoots (250 mg fresh weight) were homogenised in an extraction solvent of chloroform-methanol (v/v) with warm wa­ter. After agitation and centrifugation (10,000 g, 5 min, 4 .C), the phase that contained total lipids was collected according to Hagege et al. (1990 a). The assay was based on the oxidising properties of

Reducing Properties and Lipid Peroxidation in Hyperhydric Shoots 341

hydroperoxy-free radicals towards Fe2+ ions (Koch et al., 1958). The product of the oxidation (Fe3+ ions) reacted with ammonium thio­cyanate to make a complex that absorbs at 480 nm.

Antilipoperoxidant potential

About 250 mg of fresh shoots were ground in 10 mL cold phos­phate buffer (pH 7) in a potter and the homogenate was centrifuged (20,000 g. 10 min, 4°C). The antilipoperoxidant potential of the soluble extracts of the shoots was investigated according to Hagege et al. (1993) by measuring the capacity of 100~L supernatant to in­hibit an autooxidation cycle of linoleic acid (an emulsion of 0.2 % linoleic acid (Sigma) in 0.06 mollL phosphate buffer, pH 7.4, using the zwitterionic detergent CHAPS at 0.2%) exposed during 18h to 'Y-rays (dose of 180 Grays) in rubber-sealed vials of 10 mL. The pen­tane formed (among other volatile compounds) was measured through FIO-GC (column of porapak T; injector and detector tem­peratures: 140 and 160°C).

Hydrogm peroxitk contmt

Fresh shoots (300 mg) were frozen in liquid N2 and ground to a powder in a mortar together with cold TCA 5 % (5 mL) and acti­vated charcoal (300 mg). The extract was centrifuged (18,000 g. 10 min, 4°C). The supernatant was filtered (0.22 ~m) and the fil­trate was adjusted to pH 8.4 with 6 mollL ammonia solution. Each extract was divided into aliquots of 0.5 mL. To half of these (the blanks) 1 ~g of catalase was added. The blanks were kept at 20°C for 10 min together with the solutions without catalase, mer which 0.5 mL of colorimetric reagent prepared according to Patterson et al. (1984) was added to both series. Estimation was based on its reac­tion with a [TI(IV)] - [4-(-2-pyridylazo)resorcinol (PAR)] complex that absorbs at 508 nm.

Lipoxygtnas~ activity

Fresh shoots (300 mg) were homogenized in a prechilled mortar with 3 mL 50 mmol/L cold phosphate buffer, pH 7. Homogenate was centrifuged (18,000 g. 15 min, 4°C) and the resulting superna­tant was used as the enzyme source. The assay was done spectropho­tometrically, using linoleic acid as substrate according to Axelrod et al. (1981). The substrate was prepared by adding 50 mg of linoleic acid to 50 mg Tween 20. Sodium borate buffer (0.1 mol/L, pH 9) was progressively added (4mL) with further stirrings with a glass rod and ultrasonic dispersions. The solution was cleared by the addition of 250 ~L of NaOH 1 mol/L and made up to 25 mL as final volume with sodium borate buffer. Enzyme extract (100 ~L) was added to 2.89 mL sodium borate buffer and 10 ~L of substrate. The increase of absorbance was monitored at 234 nm.

Exprtssion of mults

Because of the hyperhydricity in one of the two materials, com­pared results were mostly expressed per unit dry weight (determined after drying, in an oven at 80°C for 48 h) or per unit proteins. Pro­teins were assayed by the coomassie blue method (Spector, 1978) with bovine serum albumin as standard.

Although the techniques used were selected on the basis of their high specificity, it can never be ascertained that contaminants with similar properties were not somehow altering the results. The re­ported determination should therefore be considered in terms of equivalent of each of the compounds assayed. The extract contents of equivalent AA, OHA, GSH, GSSG, a-tocopherol, MOA, and H20 2 were calculated using standard dose-response curves estab­lished with corresponding pure commercial compounds.

All experiments were performed on three (n = 3) or six (n = 6) separate whole shoot series without basal callus and damaged leaves. Bars in the graphs represent the standard errors.

Results

Ascorbate and dehydroascorbate (Fig. 1)

M content in NS remained stable for 3 days, decreased up to day 7 and increased later to the initial concentration. DHA content slowly decreased in NS during the first 7 days of the culture period, then it progressively increased until the end of the culture. In HS, M content was higher throughout the culture period. It reached two peaks at the 7th and 21st days and decreased at the end of the culture. DHA content was

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FagoI: Ascorbate and dehydroascorbate levels in normal (Agar) and in hyperhydrating (Gelrite) shoots during a 28-day culture period. Mean ± SO (n = 3).

342 T. FRANCK, c. KEvERS, C. PENEL, H. GREPPIN, J. F. HAUSMANN, and T. GASPAR

also higher in HS, throughout the whole culture period. Un­like AA, DHA content showed two dips at the 7th and 21st days and increased at the end of the culture. The ANDHA ratio measured was higher in NS than in HS throughout the whole culture period.

Reductd and oxidized glutathione (Fig. 2)

Reduced glutathione content slowly increased in NS and HS up to day 21, then decreased strongly afterwards. GSH content in HS was higher than in NS during the 21st day of the culture period. At the end of the culture GSH contents in both regimens were similar. Oxidized glutathione, like GSH content, increased in NS until the 21st day of the culture pe-riod, then rapidly decreased during the last week of the cul-

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ture. In HS, GSSG content increased more rapidly than in 0 -r----...,.-----,------r-----r--'

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Ftg. 2: Reduced and oxidized glutathione levels in normal (Agar) and in hyperhydrating (Gelrite) shoots during a 28-day culture pe­riod. Mean ± SD (n = 3).

o 7 14 21 28

Flg.3: ex-tocopherol level in normal (Agar) and in hyperhydrating (Gelrite) shoots during a 28-day culture period. Mean ± SD (n = 3).

NS between the 3rd and the 14th day of the subculture. GSSG content remained stable during the last 2 weeks of the culture in HS and remained higher than in NS at the end of the culture period.

Interestingly, the GSH/GSSG ratio in NS reached a peak (the 7th day) earlier than in HS (the 21st day) during the cul­ture period. At the 21st day of the culture period, the GSHI GSSG ratio was higher in HS but it strongly decreased dur­ing the last week under the ratio ofNS.

a-tocopherol (Fig. 3)

Evolution of a-tocopherol content was similar in NS and HS during the culture period. It decreased in both regimens during the first week of the culture period, then slowly in­creased until the end of the culture.

Peroxide index (Fig. 4) and malondialdehyde content (Fig. 5)

The peroxide index sharply decreased from the beginning of the culture period up to days 3 and 7 for NS and HS, re­spectively, before increasing, but it always remained inferior in the HS. MDA content decreased more rapidly in HS than in NS and remained lower than in NS throughout the cul­ture period.

Ferricyanide reduction, antilipoperoxidant potentia4 hydrogen peroxide content, and lipoxygenase activity (Table 1)

After 28 days of culture, the reduction of ferricyanide was lower in HS than in NS. HS had a high antilipoperoxidant potential in comparison to NS. H20 2 content was nearly the same in the two regimens after the experimental culture pe­riod and LOX activity was higher in HS than in NS.

Reducing Properties and Lipid Peroxidation in Hyperhydric Shoots 343

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Fig.4: Peroxide index in normal (Agar) and in hyperhydrating (Gel­rite) shoots during a 28-day culture period. Mean ± SO (n = 3).

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Fig. 5: TBA reactive substances in normal (Agar) and in hyperhyd­rating (Gelrite) shoots during a 28-day culture period. Mean ± SO (n= 3).

Discussion

AA and GSH are the two most important water-soluble antioxidants in vivo and the two major substrates of the Hal­liwell-Asada pathway. Being hydrophilic, they are principally found in the cytosol and chloroplast stroma (Winston, 1990; Meister, 1992). Generally, in plants undergoing a stress re­sponse, the enzymes of the Halliwell-Asada pathway and their main substrates have respectively higher activities and levels than those encountered under normal conditions (Elstner and Osswald, 1994; Foyer et al., 1994). In HS, supposed to be under stress, all of the enzymes of the chloroplast defense pathway were found with lower activities than in NS (see Introduction). Curiously, here we found a higher level of their substrates (AA and GSH) in HS (Figs. 1,2) and also of

Table 1: Ferricyanide reduction. antilipoperoxidant potential. H20 2 content. and lipoxygenase activity after 28 days of culture in normal (Agar) and in hyperhydrating (Gelrite) shoots. Mean ± SO (n = 6).

Normal shoots after Hyperhydric shoots after 28 days of culture 28 days of culture on agar on gelrite

Ferricyanide reduction 0.9±0.05 (.:10D/min/g FW) Antilipoperoxidant 0.1 ±0.04 potential (% inhibition of auto­oxidation cycle of linoleic acid/mg protein) H20 2 content 8.8± 1.2 ijJ.mol/mgDW) Lipoxygenase activity (2.7±0.s)X 10-3

(.:10D/mg protein)

0.49±0.03

0.34±0.05

8.2±1.8

(5.5 ± 1.3) X 10-3

DHA and GSSG. First, the general increase in HS of reduced and oxidized forms of ascorbate and glutathione might be due to the decrease of all enzyme activities that use them as substrates. The lower AAlDHA and GSH/GSSG ratios gen­erally observed in HS resulted from the low activities of the enzymes implicated in the regeneration of AA and GSH. Sec­ondly, notwithstanding the low activities of these enzymes, the regeneration of AA and GSH respectively via DHA and GSSG seems more efficient in HS. At the peaks of AA (the 7th and 21st days of the culture period) and GSH (the 21st day) contents, respectively, cortespondence of the minima of DHA and GSSG contents could be seen. These results sug­gest that HS have other possibilities to generate AA and GSH. Indeed, AA could be regenerated spontaneously by dis­proportionation of MDHA Oahnke et al., 1991) and GSH could be produced by an alternative system involving gluta­thione synthetase (Moran et al., 1994).

On the other hand, the turn-over of the Halliwell-Asada pathway depends on the reducing capability of NAD(P)H. The plasma membrane of plant cells involves redox activities that can transfer electrons from cytosolic electron donors to apoplastic electron acceptors (Crane et al., 1985). It is gener­ally accepted that NAD(P)H is the cytosolic electron donor in most cases (Malerba et al., 1995). The lower level of ferri­cyanide reduction observed in HS after 28 days of culture (Table 1) could be attributed to a general decrease of NAD (P) H content. The low activities of monodehydroascor­bate reductase and glutathione reductase observed by Franck et al. (1995) could be explained by their dependence of the reducing capacity ofNAD(P)H. NAD(P)H can be supplied via the light-driven electron transport reaction in the chloro­plast or supplied via pentose phosphate enzymes such as glu­cose-6-phosphate dehydrogenase (Polle et al., 1992). The chlorophyll decrease and the chloroplast degeneration re­cently observed in HS of Prunus avium L. (Franck et al., 1997) could explain the low production of NAD(P)H. On the other hand, a lower activity of glucose-6-phosphate de­hydrogenase has also been observed in HS of Prunus avium L. (c. Verbaere, pers. comm.). Therefore, a lack of

344 T. FRANCK, c. KEvERS, C. PENEL, H. GREPPIN,J. F. HAUSMANN, and T. GASPAR

NAD (P) H could be a limiting factor for the functioning of ~e Halliwell-Asada pathway in HS.

In addition to the low activity of the defense pathway of the chloroplast against H20 2, a decrease in catalase and per­oxidase activities and an increase of SOD activity were re­ported in HS. These results support the hypothesis of an ac­cumulation of H 20 2 in these abnormal shoots, leading to subsequent lipoperoxidation and membrane damage (Kumar and Knowles, 1993). In plants, lipid peroxidation results in an oxidative deterioration of PUFA ;md may have two ori­gins: enzymatic due to LOX activity (Axelrod et al., 1981) or autocatalytic due to activated oxygen species (Cheeseman et al., 1984). Hydroperoxides and MDA were often considered as indicators of membrane damage (Hagege et al., 1990 a, b). Hydroperoxides are the initial products of lipid oxidation and usually account for the majority of bound oxygen measured by the peroxide value. MDA and a variety of aldehydes have long been recognized as secondary products derived from the degradation of lipid hydroperoxides. Although MDA detec­tion with TBA is often used as a test for lipid rancidity (Ko­sugi and Kirugawa, 1989), it should be rather representative of the presence of aldehydic compounds and other interfering substances in the TBA reaction (Esterbauer et al., 1991; Che­rif et al., 1996). Our results show less lipid peroxidation markers throughout all of the culture period in HS (Figs. 4 and 5). The level of a-tocopherol, the most important anti­oxidant incorporated into the membrane and which may act directly as a chain breaker of lipid peroxidation (Winston, 1990), stays unchanged between HS and NS (Fig. 3). The H 20 2 content at the end of the culture period is also the same between the two types of shoots (Table 1). These results are in contradiction with the above hypotheses. A simple ex­planation would be that HS contained less active oxygen spe­cies and H20 2 responsible for membrane damage. Therefore, we should conclude that the deficiency of the enzymatic de­fense pathway in HS seems to be counterbalanced by another way of defense against these toxic elements. Indeed, the anti­lipoperoxidant potential estimation shows a higher capacity of defense in HS than in NS (Table 1). This defense should be principally ensured by AA and GSH, which can act as scavengers of reactive oxygen compounds and free radicals in the aqueous part of the cells, and can therefore indirecdy pro­tect lipid membranes from free radical chain reactions. (WInston, 1990; Meister, 1992). The fact that a-tocopherol is not implicated in the membrane defense of HS may indicate a sufficient defense by the aqueous part of the cell. This de­fense is generally not limited to AA and GSH but can also in­volve polyamines (Hagege et al., 1990 a; Borrell et al., 1997), phenols, carotenoids and flavonoids (Foyer et al., 1994) and annexin-like proteins (Gidrol et al., 1996). Polyamines al­ready have been shown to be at a higher level in hyperhydric tissues (Kevers et al., 1997). Next investigations will evaluate the other antioxidant compounds in NS and HS.

However, abnormal morphology, less chlorophyll content, chloroplast degeneration (Franck et al., 1997) and necroses of the apices (Kataeva et al., 1991) observed in HS seem to im­plicate oxidative attacks and peroxidative damage. The high activity of LOX measured in HS (Table 1) is in agreement with peroxidative damage. The PUFA can be oxidized by LOX to generate oxy-free radical forms and hydroperoxides

(Kumar and Knowles, 1993). As discussed earlier, HS have a high capacity to react direcdy with active oxygen species. However, an accumulation of hydroperoxides is not con­firmed by peroxide and MDA contents. This point is intrigu­ing. Recent studies involve the catabolism of PUFA and hy­droperoxides into compounds with a possible protective and regulating role under stress conditions (BIee and Joyard, 1996; Marechal et al., 1997). Another explanation for the low peroxide content in HS might be attributed to a decrease of cell membrane PUFA as already postulated by Arbillot et al. (1991) for habituation and by El-Sheekh and Rady (1995) for cold stress. For Dianzani (1989) the permeability and the ri­gidity of the membrane increase in response to a decrease of PUFA. A possible increase of the membrane permeability in hyperhydric tissues has already been argued by Kevers and Gaspar (1986).

Another hypothesis is that micropropagated HS of Prunus avium can be considered as rejuvenated (Hammat and Grant, 1993) under the effect or the accumulation of cytokinins, which have been shown to retard senescence and to reduce the increase in lipid peroxidation level (Hung and Kao, 1997).

As conclusion, the concept of oxidative stress as described by Lichtenthaler (1996) in relation to hyperhydricity is still not easy to determine accurately. In spite of high efficient scavenging properties (antioxidant and antilipoperoxidant), HS unavoidably progress towards abnormal injuries. Such pa­radoxical results have already been discussed in habituated and hyperhydric sugarbeet calli (Gaspar et al., 1995; Hagege, 1996).

Acknowledgements

This research was supported by the «Region Wallo nne» through the Prime contract (30108) provided to CEDEVIT. T. F. gratefully acknowledges the Luxembourg Ministry of National Education for supporting this investigation at the University of Liege and a grant from the Fonds National de la Recherche Scientifique that allowed him to stay at the University of Geneve.

References

ARBILLOT, J., J. LE SAOS, J. P. BILLARD, J. BOUCAUD, and T. GASPAR: Changes in fatty acid and lipid composition in normal and habi­tuated sugar beet calli. Phytochem. 30, 491-494 (1991).

AsAoA, K.: Ascorbate peroxidase - a hydrogen peroxide - scavenging enzyme in plants. Physiol. Plant. 85,235-241 (1992).

AsAoA, K. and M. TAKAHASHI: Production and scavenging of active oxygen in photosynthesis. In: KYLE, D. J., C. B. OSMOND, and C. J. ARNTzEN (eds.): Photoinhibition, pp. 227-2ff7. Elsevier Science Publishers (1987).

AxELROD, B., T. M. CHEESBROUGH, and S. LAAKso: Lipoxygenase from soybeans. Methods in Enzymology 71, 441-451 (1981).

BLEE, E. and J. JOYARD: Envelope membranes from spinach chloro­plasts are a site of metabolism of fatty acid hydroperoxides. Plant Physiol. 110,445-454 (1996).

BORRELL, A., L. CARBONELL, R. FARR.\s, P. PUIG-PARELLADA, and A. F. TIBURCIO: Polyamines inhibit lipid peroxidation in senescing oat leaves. Physiol. Plant. 99, 385-390 (1997).

CARRIi, B., T. GASPAR, H. GREPPIN, and C. PENEL: Redox charac­teristics of normal and habituated cell lines of sugarbeet. Plant Cell and Environment 17, 457-461 (1994).

Reducing Properties and Lipid Peroxidation in Hyperhydric Shoots 345

CHEESEMAN, K. H., G. W BURTON, K. U. INGOLD, and T. E SLA­TER: Lipid peroxidation and lipid antioxidants in normal and tu­mor cells. Toxicologic Pathology 12, 235-239 (1984).

CHERIF, M., P. NODET, and D. HAGEGE: Malondialdehyde cannot be related to lipoperoxidation in habituated sugarbeet plant cells. Phytochemistry 41, 1523-1526 (1996).

CRANE, E L., H. SUN, M. G. CLARK, G. GREBING, and H. LOw: Transplasma membrane redox systems in growth and develop­ment. Biochim. Biophys. Acta 811, 233-264 (1985).

DEBERGH, P., J. AITKEN-CHRISTIE, D. COHEN, B. GROUT, S. Vo'N ARNOLD, R. ZIMMERMAN, and M. ZlV: Reconsideration of the term vitrification as used in micropropagation. Plant Cell Tissue Organ Cult. 30, 140-165 (1992).

DlANZANI, M. U.: Lipid peroxidation and cancer: a critical reconsi­deration. Tumori 75,351-357 (1989).

EL-SHEEKH, M. M. and A. A. RAoY: Temperature shift-induced changes in the antioxidant enzyme system of cyanobacterium Synechocystis PCC 6803. Biologia Plant. 37, 21-25 (1995).

ELSTNER, E. E and W OSSWALD: Mechanisms of oxygen activation during plant stress. Proceedings of the Royal Society of Edin­burgh 102B, 131-154 (1994).

EsTERBAUER, H., R. J. SCHAUR, and H. ZOLLNER: Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Rad. BioI. Med. 11, 81 (1991).

FOYER, c. H. and B. HALLIWELL: The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133,21-25 (1976).

FOYER, c. H., M. LELANDAlS, and K. J. KUNERT: Photooxidative stress in plants. Physiol. Plant. 92,696-717 (1994).

FRANCK, T., c. KEVERS, and T. GASPAR: Protective enzymatic sys­tems against activated oxygen species compared in normal and hyperhydric shoots of Prunus avium L. raised in vitro. Plant Growth Reg. 16, 253-256 (1995).

FRANCK, T., M. CRilVECOEUR, J. WUEST, H. GREPPIN, and T. GAS­PAR: Cytological comparison of leaves and stems of Prunus avium L. shoots cultured on a solid medium with agar or gelrite. Bio­technic and Histochemistry 73, 32-43 (1997).

GASPAR, T.: Vitrification in micropropagation. In: BAJAJ, Y. P. S. (ed.): Biotechnology in Agriculture and Forestry, High-Tech and Micropropagation I, Vol. 17, pp. 117-126. Springer-Verlag, Berlin (1991).

GASPAR, T., c. KEVERS, T. FRANCK, B. BISBIS, J. P. BILLARD, C. HUAULT, E LE DILY, G. PETlT-PALY, M. RIDEAU, C. PENEL, M. CRilVECOEUR, and H. GREPPIN: Paradoxical results in the analysis of hyperhydric tissues considered as being under stress: questions for a debate. Bulg. J. Plant Physiol. 21, 80-97 (1995).

GAVIDIA, I., c. ZARAGOZA, J. SEGURA, and P. PEREZ-BERMUDEZ: Plant regeneration from juvenile and adult Anthyllis cytisoities, a multipurpose leguminous shrub. J. Plant Physiol. 150,714-718 (1997).

GIDROL, X., P. A. SABELU, Y. S. FERN, and A K. KUSH: Annexin­like protein from Arabidopsis thaliana rescues floxyR mutant of Escherichia coli from H 20 2 stress. Proc. Nat!. Acad. Sci. 93, 11268-11273 (1996).

HAGEGE, D.: Habituation in sugarbeet plant cells: Permanent stress or antioxidant adaptative strategy? In Vitro Cell. Dev. BioI. Plant 32, 1-5 (1996).

HAGEGE, D., c. DEBY, C. KEVERS, J. M. FOlDART, and T. GASPAR: Antilipoperoxidant potential of the fully habituated nonorgano­genic sugarbeet callus. Arch. Int. Physiol. Biochim. Biophys. 101, 9 (1993).

HAGEGE, D., c. KEVERS, J. BOUCAUD, M. DUYME, and T. GASPAR: Polyarnines, phospholipids and peroxides in normal and habi­tuated sugar beet calli. J. Plant Physiol. 136, 641-645 (1990a).

HAGEGE, D., A. NOUVELOT, J. BOUCAUD, and T. GASPAR: Malon­dialdehyde titration with thiobarbiturate in plant extracts: avoi­dance of pigment interference. Phytochem. Anal. 1, 86-89 (1990 b).

HAMMAT, N. and N. J. GRANT: Apparent rejuvenation of mature wild cherry (Prunus avium L.) during micropropagation. J. Plant Physiol. 141, 341-346 (1993).

HUNG, K. T. and C. H. KAo: Lipid peroxidation in relation to se­nescence of maize leaves. J. Plant Physiol. 150,283-286 (1997).

JAHNKE, L. S., M. R. HULL, and S. P. loNG: Chilling stress and oxy­gen metabolizing enzyme in Zea mays and Zea dip/operennis. Plant Cell and Environment 14, 97-104 (1991).

KATAEVA, N. v., I. G. ALExANDROVA, R. G. BUTENKO, and E. V. DRAGAVTCEVA: Effect of applied and internal hormones on vitri­fication and apical necrosis of different plants cultured in vitro. Plant Cell Tissue Organ Cult. 27, 149-154 (1991).

KEVERS, c., B. BISBIS, T. FRANCK, E LE DILY, C. HUAULT, C. BIL­LARD, J. M. FOlDART, and T. GASPAR: On the possible causes of polyamine accumulation in in vitro plant tissues under neoplasic progression. In: GREPPIN, H., C. PENEL, and P. SIMON (eds.): Travelling Shot on Plant Development, pp. 63-71. Univ. of Ge­neva, Switzerland (1997).

KEVERS, c., M. COUMANS, M. E COUMANS-GILLES, and T. GASPAR: Physiological and biochemical events leading to vitrification of plants cultured in vitro. Physiol. Plant. 61, 69-74 (1984).

KEVERS, C. and T. GASPAR: Vitrification of carnation in vitro: changes in water content, extracellular space, air volume, and ions levels. Physiol. Veg. 24, 647-653 (1986).

KOCH, R. B., B. STERN, and C. G. FERRARI: Linoleic acid and trili­nolein as substrate for soybean lipoxidase(s). Arch. Biochem. Bio­phys. 78, 165-179 (1958).

KOSUGI, H. and K. KIRUGAWA: Potential thiobarbituric acid-reactive substances in peroxidized lipids. In: Free Radical Biology and Medicine, vol. 7, pp. 205-207. Maxwell Pergamon Macmillan pic (1989).

KUMAR, G. N. M. and N. R. KNOWLES: Changes in lipid peroxida­tion and lipolytic and free-radical scavenging enzyme activities during aging and sprouting of potato (Solanum tuberosum) seed­tubers. Plant Physiol. 102, 115-124 (1993).

LICHTENTHALER, H. K.: Vegetation stress: an introduction to the stress concept in plants. J. Plant Physiol. 148,4-14 (1996).

MALERBA, M., P. CROSTl, and R. BlANCHETTl: Ferricyanide induced ethylene production is a plasma membrane proton pump de­pendent 1-aminocydopropane-1-carboxylic acid (ACC) oxidase activation. J. Plant Physiol. 147, 182-190 (1995).

MAREcHAL, E., M. A. BLOCK, A. J. DORNE, R. DOUCE, and J. Jo­YARD: Lipid synthesis and metabolism in the plastid envelope. Physiol. Plant. 100, 65-77 (1997).

MEISTER, A.: On the antioxidant effects of ascorbic acid and gluta­thione. Bioch. Pharm. 44, 1905-1915 (1992).

MORAN, J. E, M. BECANA, I. ITURBE-ORMAETXE, S. FRECHILLA, R. V. KLUCAS, and P. APARICIo-TEJO: Drought induces oxidative stress in pea plants. Planta 194, 346-352 (1994).

PATTERSON, B. D., E. A MACRAE, and I. B. FERGUSON: Estimation of hydrogen peroxide in plant extracts using titanium(lV). Ana­lytical Biochemistry 139, 487-492 (1984).

POLLE, A, K. CHAKRABARTl, S. CHAKRABARTl, E SEIFERT, P. SCHRA­MEL, and H. RENNENBERG: Antioxidants and manganese defi­ciency in needles of Norway spruce (Picea abies L.) trees. Plant Physiol. 99, 1084-1089 (1992).

SANKHLA, D., S. TRIVEDI, T. D. DAVIS, and N. SANKHLA: Toxic oxy­gen accumulation and hyperhydricity. In Vitro Cell. Dev. BioI. 30A Pt. II, 75 (1994).

SPECTOR, T.: Refinement of the coomassie blue method of protein quantitation. Anal. Biochem. 86, 142-146 (1978).

346 T. FRANCK, c.1<BvERs, C. PENEL, H. GRBPPIN, J. F. HAUSMANN, and T. GASPAR

UNDENFRIBND, S.: Fluorescence Assay in Biology and Medicine, pp. m. Academic Press, London (1962).

WANG, S. Y., H. J. JIAO, and M. FAUST: Changes in ascorbate, gluta­thione, and rdated enzyme activities during thidiazuron-induced bud break of apple. Physiol. Plant. 82,231-236 (1991).

WINSTON, G. w.: Physicochemical basis for free radical formation in cells: production and defenses. In: ALsCHER, R G. and J. R

CUMMING (eds.): Stress Responses in Plants: Adaptation and Ac­climation Mechanisms, pp. 57-86. Wuey-Liss (1990).

Zrv~ M.: Vitrification: morphological and physiological disorders of in vitro plants. In: DEBERGH, P. C. and R H. ZIMMERMAN (eds.): Micropropagation. Technology and Applications, pp. 45-69. Kluwer Academic Publishers, Dordrecht (1991).