Ascorbate and glutathione metabolism during ... - CSIRO Publishing

CSIRO PUBLISHING

Functional Plant Biology, 2007, 34, 601–613

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Ascorbate and glutathione metabolism during development and desiccation of orthodox and recalcitrant seeds of the genus Acer Stanislawa PukackaA,B and Ewelina RatajczakA A

Seed Biochemistry Laboratory, Institute of Dendrology, Polish Academy of Sciences, ´ 62-035 Kornik, Poland. B Corresponding author. Email: [email protected]

Abstract. The ascorbate–glutathione system was studied during development and desiccation of seeds of two Acer species differing in desiccation tolerance: Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant). The results showed remarkable differences in the concentration and redox balance of ascorbate and glutathione between these two kinds of seeds during development, and a significant dependence between glutathione content and acquisition of desiccation tolerance in Norway maple seeds. There were relatively small differences between the species in the activities of enzymes of the ascorbate–glutathione cycle: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2). At the end of seed maturation, ascorbic acid content and the activities of the above enzymes was about the same in both species The electrophoretic pattern of APX isoenzymes was also similar for both species, and the intensity of the bands decreased at the end of seed maturation in both species. When sycamore seeds were desiccated to a moisture content of less than 26%, there was a marked decrease in seed viability and an increase in the production of reactive oxygen species. During desiccation, Norway maple seeds had a more active defence system, which was reflected in a higher glutathione content, a higher glutathione redox status, a higher ascorbate redox status, and higher activities of APX, MR, DHAR, GR and GPX (glutathione peroxidase). During desiccation, sulfhydryl-to-disulfide transition into proteins was more intense in Norway maple seeds than sycamore seeds. All of these results suggest that, in orthodox seeds, the ascorbate–glutathione cycle plays an important role in the acquisition of tolerance to desiccation, in protein maturation, and in protection from reactive oxygen species. Additional keywords: ascorbic acid, ascorbate peroxidase, dehydroascorbate reducrase, glutathione reductase, monodehydroascorbate reductase, redox balance.

Introduction The Norway maple (Acer platanoides) and the sycamore (Acer pseudoplatanus) belong to the same genus, and grow under similar climatic conditions; nevertheless, their seeds differ in terms of tolerance to dessication. In Poland, Norway maple seeds and sycamore seeds usually reach full maturity at the end of September, and are shed in early October. However, Norway maple trees and sycamore trees flower at different times (Pukacka and Pukacki 1997; Pukacka 1998). They also differ in the dynamics of seed development. The Norway maple usually starts to flower 3 weeks earlier than the sycamore but embryo formation starts earlier in the sycamore (Pukacka 1998). However, the dry weight of seeds peaks at about the same time in both species. Norway maple seeds are tolerant to desiccation, whereas sycamore seeds are classified as recalcitrant (Hong and Ellis 1990; Dickie et al. 1991). Norway maple seeds become tolerant to desiccation 18 weeks after flowering (WAF) (Pukacka and Pukacki 1997; Pukacka and Wójkiewicz 2002). After that © CSIRO 2007

time, they can be slowly desiccated at ambient temperature until they reach moisture content (MC) of 8–10%, without loss of viability. They are typical orthodox seeds. Before they are shed at the end of maturation, their moisture content can be as low as 27%. In contrast, sycamore seeds remain sensitive to desiccation throughout development period (Dickie et al. 1991), and dehydration below 27% MC leads to a significant decrease in seed germinability (Pukacka and Czubak 1998). However, there is some evidence that viability is retained to 15% MC (Jahnel 1955). Recently, Daws et al. (2006) found that the desiccation tolerance of sycamore seeds depends on the heat sum during development, so the tolerance of seed lots from France or Italy is the more consistent with the intermediate category than with the recalcitrant category. To the end of maturation sycamore seeds maintain a high MC of 50–55% (Pukacka 1998). Seed development can be divided into three stages: embryogenesis, seed filling (cell enlargement and reserve deposition), and maturation. Seed development starts with a

10.1071/FP07013

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Functional Plant Biology

zygotic embryo and ends with a mature seed that is capable of germination. During seed development, the metabolic activity and mitochondrial respiration rate are high, which suggest that a significant amount of reactive oxygen species (ROS) can be generated. The level of ROS is under the strict control of antioxidant mechanisms, prevented undesirable, destructive effects of their activity. However, ROS perform important roles as signalling molecules regulating growth and development and coordinating responses to abiotic and biotic stress (Desikan et al. 2005). The ascorbate system may play a central role in embryogenesis and seed filling (Arrigoni et al. 1992; De Gara et al. 2003; De Tullio and Arrigoni 2003). Ascorbic acid (ASA) may affect the progression of the cell-cycle (Noctor and Foyer 1998; Potters et al. 2002). ASA may also affect cell growth by modulating the expression of genes involved in hormonal signalling pathways (Pastori et al. 2003). In developing seeds, ASA modulates the synthesis of plant hormones, such as ethylene, GA and ABA. In this way, ASA indirectly affects seed growth. Most studies of the role of ROS in seed development have focused on how the seeds acquire tolerance to desiccation. In orthodox seeds, the acquisition of tolerance to desiccation can be compared to defence mechanisms in dehydrated vegetative tissues in which antioxidant systems play a crucial role. The ascorbate–glutathione (A–G) cycle is an important pathway of indirect or direct scavenging ROS in cells (Noctor and Foyer 1998; Foyer and Noctor 2003). Earlier research confirmed its role in plant defence reactions to many abiotic stress factors, including desiccation (Allen et al. 1997; Foyer et al. 1997). Ascorbate peroxidase (APX) is an important enzyme participating in cell detoxification because of its presence in all cell compartments and its high affinity to hydrogen peroxide (H2 O2 ). The electron donor is ASA, whose pool is regenerated by monodehydroascorbate reductase (MR) and dehydroascorbate reductase (DHAR), with the participation of reduced glutathione (GSH) and NADPH. GSH is oxidised to GSSG and reduced back to GSH by glutathione reductase (GR). Besides participating in A–G cycle, ASA can react directly with hydroxyl radicals, superoxide anion radicals, and single oxygen (Noctor and Foyer 1998). ASA is also a secondary antioxidant, and reduces the oxidized form of α-tocopherol. α-tocopherol is an important antioxidant associated with cell membranes (Padh 1990). Tommasi et al. (1999) showed differences in ascorbate content and ascorbate-related enzymes such as APX, MR and DHAR between orthodox (Pinus pinea L., Vicia faba L., Avena sativa L.) and recalcitrant seeds (Ginkgo biloba L., Aesculus hippocastanum L., Quercus cerris L. and Cycas revoluta Thunb.) before and after dehydration. Recalcitrant seeds were characterised by high ascorbate content and high activity of enzymes of the ascorbate system compared with orthodox seeds, where ASA content and APX activity significantly diminished or totally disappeared during maturation and desiccation. Glutathione is the major antioxidant and redox buffer in the cell (Schafer and Beuttner 2001). In response to oxidative stress, GSH is converted to GSSG which changes the redox status of the cell. Kranner and Grill (1993) observed accumulation of GSSG in pea seeds in the dry state. Greggains et al. (2000) found that during desiccation, Norway maple seeds

S. Pukacka and E. Ratajczak

contained higher levels of total glutathione than sycamore seeds. The involvement of glutathione redox status in withstanding desiccation by orthodox seeds has been little investigated during acquisition of desiccation tolerance. Kranner et al. (2006) supposed that glutathione half-cell reduction potential is an important marker of lethal changes in plant cells (including seed cells during ageing and desiccation) caused by oxidative stress. In this paper we compare the whole complex of reactions of components of the A–G cycle during development, maturation and desiccation of seeds in two species that differ in desiccation tolerance but belong to the same genus and occur in similar climatic conditions. The aim of this study was to show the role of this system in seed development and acquisition of desiccation tolerance. Materials and methods Plant material The seeds were collected from single trees of Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.) in the Kórnik Arboretum (Poland) during the growing seasons of 2004 and 2005. Seeds were collected at 2 week intervals, beginning at 11 WAF for sycamore and 14 WAF for Norway maple. After harvest, the seeds were transported immediately to the laboratory in sealed plastic bags. At each harvest of the Acer tree, three samples of 20 freshly harvested seeds, extracted from the fruit and the seed coat were used for MC determination after drying at 120◦ C for 24 h. Tolerance to desiccation of Norway maple seeds during development Tolerance to desiccation was determined by testing germination as described by Pukacka and Pukacki (1997). Results were confirmed by testing ionic conductance. Each week, seed samples were collected and desiccated at ambient temperature until the moisture content was between 10 and 12%. After careful rehydration at a relative humidity (RH) of 100%, the seeds were stratified for 16–20 weeks at 3◦ C. (Pukacka and Pukacki 1997). After several weeks of cold stratification, the seeds that were not damaged during desiccation started to germinate. Germination (radicle emergence) of seeds was checked at weekly intervals, and decayed and germinated seeds were removed. For the conductance test, three samples of 20 desiccated seeds were placed in 10 mL of deionised water. After 24 h of incubation at ambient temperature, the ionic conductance was measured and recorded in mS g−1 DW. Desiccation of seeds Mature seeds were collected 24 WAF for Norway maple and 21 WAF for sycamore. The seeds were desiccated at 18–20◦ C and 60–70% RH. Sycamore seeds were removed from the samaras before desiccation. Moisture content was measured as described above. Ascorbate assays Ascorbic acid (ASA) and dehydroascorbate (DHA) content were measured using the procedure described by Kampfenkel et al. (1995). The assay is based on the reduction of Fe3+ to Fe2+ by ascorbic acid in acidic solution. Fe2+ forms complexes with

Ascorbate and glutathione metabolism in Acer seeds

bipirydyl, yielding a pink colour with the absorbance peak at 525 nm. Each measurement was made using three samples of 50 embryonic axes or 10 cotyledons. The samples were homogenised in cold 6% TCA (w/v). The homogenate was centrifuged for 10 min at 12 000g, and the supernatant was used for the assay. The data recorded represent the means and standard deviations of two measurements with three different extracts. Glutathione assays Glutathione in the reduced (GSH) and the oxidised form (GSSG) was determined according to Smith (1985). Samples of 50 embryocic axes or 10 cotyledons each were homogenised in 5% (w/v) sulfosalicylic acid on ice bath and then centrifuged at 10 000g for 20 min. A 1-mL aliquot of the supernatant was removed and neutralised by adding 1.5 mL of 0.5 M Kphosphate buffer pH 7.5. This sample was used for the determination of total glutathione (GSH + GSSG). Another 1 mL of neutralised supernatant was pretreated with 0.2 mL of 2-vinylpyridine for 1.5 h at 25◦ C to mask GSH and to allow determination of GSSG alone. Both samples were extracted twice with 5 mL diethylether. The reaction mixture contained: 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.2 mL of 6 mM 5,5 -dithiobis-(2nitrobenzoic acid), 0.1 mL of 2 mM NADPH, 0.1 mL (1 unit) of glutathione reductase type III (Sigma-Aldrich, Poland), and 0.1 mL of extract. The change in absorbance at 412 nm was followed at 25◦ C. A standard curve was prepared by using the GSH (Sigma-Aldrich) standard. Results are expressed as means (± s.d.) of two measurements conducted with three different extracts. Enzyme extraction and assays All extraction procedures were conducted at 4◦ C. Samples of 50 embryonic axes or 10 cotyledons each were ground in liquid nitrogen and homogenised in 50 mM sodium phosphate buffer, pH 7.0, containing 0.2 mM EDTA and 20% polyvinylpolypyrrolidone (PVPP) for 10 min. Homogenates were filtered through two layers of cheesecloth and centrifuged at 4◦ C at 15 000g for 20 min. The supernatant was desalted on a Sephadex G25 (Sigma-Aldrich) column according to Helmerhorst and Stokes (1980). Ascorbate peroxidase (APX, EC.1.11.1.11) activity was measured by following the decrease in absorbance at 290 nm owing to ASA oxidation for 5–10 min, according to Nakano and Asada (1981). The reaction mixture contained: 1 mL of 0.68 mM ASA and 0.1 mM EDTA in 0.1 M phosphate buffer pH 7.0, 1 mL of 4 mM H2 O2 and 50–100 µL of the enzyme extract. APX activity was expressed as nmoles ASA min−1 mg−1 protein. Monodehydroascorbate reductase (MR, EC 1.6.5.4) was tested according to Zhang and Kirkham (1996). The reaction mixture contained: 50 mM phosphate buffer pH 7.6, 0.1 mM NADH, 2.5 mM ASA, four units of ASA oxidase (SigmaAldrich) and 50 µL of enzyme extract. NADH oxidation was monitored at 340 nm. MR activity was expressed as nmoles NADH min−1 mg−1 protein. Dehydroascorbate reductase (DHAR, EC. 1.8.5.1) activity was measured according to Arrigoni et al. (1992). The reaction mixture contained: 0.9 mL of 0.05 M potassium phosphate buffer pH 6.3, 100 µL of 13.5 mM dehydroascorbate (DHA), 100 µL of


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13.5 mM reduced glutathione (GSH) and 100–200 µL of enzyme extract. The DHAR activity was monitored by following the formation of ascorbate at 265 nm for 5 min. Enzyme activity was expressed as nmoles ASA min−1 mg −1 protein. Glutathione reductase (GR, EC. 1.6.4.2) activity was determined according to Esterbauer and Grill (1978), by following the rate of NADPH oxidation at 340 nm. The assay mixture contained: 0.5 mM NADPH, 10 mM oxidised glutathione (GSSG), 10 mM EDTA in 0.1 M phosphate buffer pH 7.8, and 50–100 µL of the enzyme extract. GR activity was expressed as nmoles NADPH min−1 mg−1 protein. Glutathione peroxidase (GPX, EC. 1.11.1.9) assay was conducted as described by Nagalakshmi and Prasad (2001). The reaction mixture contained 100 µL each of 0.5 M K phosphate buffer, pH 8.2, 10 mM EDTA, 1.14 M NaCl, 10 mM GSH, 2 mM NADPH, 2.5 mM H2 O2 . The reaction was started by adding two units of glutathione reductase, 344 µL of water, and 100 µL of enzyme extract. The disappearance of NADPH was monitored at 340 nm for 15 min. GPX activity was expressed as nmoles of NADPH min−1 mg−1 protein. The results correspond to the means ± s.d. of the values obtained with three different extracts and two measurements per extract (i.e. six measurements). Superoxide determination The production of superoxide anion radicals was determined using the procedure described by Doke (1983). The reduction of nitroblue tetrazolium was monitored in the dark at room temperature. The production of superoxide anion radicals was determined by measuring the absorbance of the end product . 530 nm. O2 − formation was expressed as A530 g −1 DW of the sample. Six samples of five seeds each were analysed. Hydrogen peroxide content Hydrogen peroxide content was determined using the ferrithiocyanate method described by Sagisaka (1976). Samples of five seeds were finely ground in liquid nitrogen. The powder was then homogenised in 5 mL of 5% TCA containing 10 mmol EDTA. The homogenate was filtered through two layers of cheesecloth and centrifuged at 12 000g for 15 min at 4◦ C. The total volume of supernatant was analysed. Total, protein bound and non-protein thiol estimation Thiol content was determined according to Sedlak and Lindsay (1968), using Elman’s reagent [5,5 -dithio-bis-(2-nitrobenzoic acid)]. Samples of 20 embryos were homogenised in 0.02 M EDTA on ice and centrifuged at 4◦ C at 20 000g for 20 min. Total thiols were estimated in supernatants. For determination of nonprotein thiols 5 mL of the supernatants were mixed with 4 mL of deionized water and 1 mL of 50% TCA. After 15 min the tubes were centrifuged at 10 000g for 15 min. The protein bound thiols were calculated by subtracting the non-protein thiols from total thiols. A standard curve was prepared using glutathione (GSH), and thiol contents were expressed as glutathione equivalents. Native PAGE analysis of APX and DHA-reducing proteins Native PAGE was performed on protein extracts from embryonic axes and cotyledons isolated at an early stage (14 WAF for


Analysis of proteic–SH groups The visualisation of proteic–SH groups was performed by labelling with mBBr according to Kobrehel et al. (1992), as described by De Gara et al. (2003). Samples (0.5 g) of embryonic axes and cotyledons were ground in liquid nitrogen with a mortar and pestle, and the next 2 mL of 2 mM mBBr (dissolved in acetonitrile) in 100 mM Tris–HCl buffer pH 7.5 were added. The thawed mixture was then ground for 1 min, transferred to a microfuge tube and centrifuged at 26 000g, at 4◦ C for 20 min. To 80 µL of supernatant, 10 µL each of 10% SDS and 100 mM 2-mercaptoethanol were added to stop the reaction and derivatise excess mBBr. SDS–PAGE analysis of mBBr-labelled extracts was performed in 12.5% gels at pH 8.5. In each lane 30 µg of the total amount of protein was loaded. After electrophoresis the gels were placed in 12% TCA for 30 min, and then were transferred to a solution of 40% ethanol (v/v) in 10% acetic acid (v/v) for 8–19 h. The fluorescence of proteins bound to mBBr was visualised in UV light (365 nm). The protein content of crude enzyme extracts was estimated according to Bradford (1976), by using BSA as a standard. Statistical analysis Data are presented as means ± s.d. of six or three replicates. The statistical differences were tested using an analysis of variance (ANOVA). Levels of significance are indicated: *P < 0.05; **P < 0.01.

Results Seed development Seed collection started 14 WAF for Norway maple, and 11 WAF for sycamore. At this time, the embryos were large enough to isolate the embryonic axes from the cotyledons so that they could be analysed separately. Seed development was observed until 24 and 21 WAF, respectively, when they were fully mature and orthodox Norway maple seeds already differed from the recalcitrant sycamore with respect to MC (Fig. 1). Starting 20 WAF, Norway maple seeds lost water more quickly, particularly in cotyledons, than sycamore seeds. By 18 WAF, Norway maple seeds had become tolerant to desiccation. This was confirmed by the germination test and also by the conductance test after drying (Fig. 2). In embryonic axes, ASA and DHA contents were about the same in both species. Redox status was also about the same. In cotyledons, levels of both ASA and DHA gradually decreased during seed maturation. The decrease was large for Norway maple seeds, but only small for sycamore seeds (Fig. 3). At the end of seed maturation, ASA content of embryonic

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A. platanoides and 11 WAF for A. pseudoplatanus) and at a late stage of seed development (22 and 19 WAF, respectively) and during desiccation. Embryonic axes and cotyledons were ground as described above. The homogenates were centrifuged at 30 000g for 30 min and the supernatants were utilised for the electrophoretic analyses. Native PAGE was performed by a Mini Protean 3 Cell (Bio-Rad, Hercules, CA) apparatus in 10 × 12 cm slab gels. A stacking gel containing 3.0% acrylamide and separating gel containing 7.5% acrylamide were used with a running buffer of 4 mM Tris–HCl pH 8.3, and 38 mM glycine. The same amount of total protein as determined by Bradford assay (Bradford 1976) was loaded in each lane. In-gel APX staining was performed according to Lee and Lee (2000). A running buffer additionally contained 2 mM ASA. After electrophoresis the gels were equilibrated with 50 mM Na-phosphate buffer (pH 7.0) containing 2 mM ASA for 30 min, and then incubated in the same buffer and 4 mM ASA and 2 mM H2 O2 for 20 min. H2 O2 was added to the solution just before the gel incubation. Afterwards, the gels were washed with 50 mM Na-phosphate buffer (pH 7.8), 28 mM TEMED and 2.4 mM NBT for 10–20 min with gentle agitation, and stopped by a brief wash with distilled water. In-gel DHA-reducing proteins (DHAR) were stained according to De Gara et al. (2000). After electrophoresis the gels were incubated with 0.1 M Na-phosphate buffer (pH 6.2), containing 4 mM GSH and 2 mM DHA, under agitation, for 10 min. The gels were then washed with distilled water and specifically stained for DHAR activity by incubation for 15 min in 0.1 N HCl containing 0.1% (w/v) ferrichloride and 0.1% (w/v) ferricyanide, which gives a colour reaction with ASA.


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axes and cotyledons of orthodox seeds (12.3 and 5.4 µmol g−1 DW, respectively) did not differ significantly from those of recalcitrant seeds (13.1 and 6.9 µmol g−1 DW, respectively). Values of glutathione content differed more markedly. An interesting relationship was observed both in embryonic axes and in cotyledons of orthodox seeds. GSH and GSSG levels clearly increased from the time of acquisition of desiccation 50

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tolerance (18 WAF) and were high until the end of seed maturation (Fig. 4). In seeds of sycamore, GSH content of cotyledons and embryonic axes decreased throughout the period of seed maturation. However, the total glutathione content (GSH + GSSG) of embryonic axes did not change until 17 WAF and started to decrease rapidly as late as 19 WAF. At the end of seed maturation, GSH content of orthodox seeds was markedly higher than that of recalcitrant seeds (2502 v. 1485 nmol g−1 DW in embryoonic axes and 2723 v. 1135 nmol g−1 DW in cotyledons of orthodox v. recalcitrant seeds, respectively). Throughout the maturation process, redox status as represented by the GSH : GSSG ratio was twice as high in embryonic axes of Norway maple as in embryonic axes of sycamore, and three times as high in cotyledons of Norway maple as in cotyledons of sycamore (Fig. 4). The patterns of activities of APX, DHAR, MR and GR were similar in both species. The activities of all of these enzymes were higher at the beginning of the observation period, probably because embryogenesis had not yet finished. Later, enzyme activities stabilised at a lower level until the end of seed maturation. Enzyme activities were generally higher in embryonic axes than in cotyledons, and from 15 to 30% higher in Norway maple seeds than in sycamore seeds (Fig. 5). APX isoforms were similar in Norway maple and sycamore seeds (Fig. 6). In both species APX activity was high in the early stage of maturation. This was determined using the spectrophotometric assay, and confirmed using native PAGE. In the late stage of maturation APX activity was lower. Some isoform bands were less intense, and others disappeared entirely. 50

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Weeks after flowering Fig. 4. Changes in the contents of reduced (GSH) and oxidized (GSSG) forms of glutathione and GSH/GSSG ratio in embryo axes and cotyledons of A. platanoides and A. pseudoplatanus seeds during development. Data are means of six replicates ± s.d.

Seed desiccation Desiccation of seeds was performed in laboratory conditions identical for both kinds of seeds. At the start of the experiment, the MC of seeds of Norway maple and sycamore were 56–52%, and 56–60%, respectively. For Norway maple seeds, there were no changes in germination rate and electrolyte leakage during desiccations. For sycamore seeds, the germination rate dropped dramatically when the seeds were desiccated to a moisture content of less than 29% (Fig. 7). Electrolyte leakage also increased from 10 to 40%. In sycamore seeds the level of superoxide anion radical . (O2 − ) markedly increased during desiccation and it was significantly higher than in Norway maple seeds. Also the level of hydrogen peroxide was higher in sycamore seeds with the exception of the last step of desiccation, where the level of H2 O2 was higher in Norway maple seeds (Fig. 8). ASA content in embryonic axes and cotyledons was similar in both species at all stages of dessication. DHA content, on the other hand, was significantly lower for Norway maple seeds than for sycamore seeds (Fig. 9). During desiccation of Norway maple seeds the GSH content significantly increased from 4.2 to 8.5 nmol per axis during the first stage of desiccation from 52 to 48% MC. This high level was maintained until the last stage (7% MC), when it decreased to 3.2 nmol per axis. Simultaneously, during this stage GSSG level almost doubled, from 2.8 to 5.8 nmol per axis (Fig. 10). In cotyledons, GSH content increased

from 36 to 102 nmol per cotyledon during the first stage of desiccation, and decreased during subsequent stages of desiccations. During all stages of desiccation, GSH and GSSG levels were significantly higher for Norway maple seeds than for sycamore seeds. The redox status of ascorbate and glutathione was significantly higher in embryonic axes and cotyledons of Norway maple seeds than in those of sycamore seeds. This was generally true during all stages of desiccations (Figs 9, 10). The assay of the activities of enzymes of the A–G cycle (APX, MR, DHAR and GR) revealed a similar trend of changes during desiccation stages. A significant increase in the activity of these enzymes, compared with the no desiccated control, was observed in embryonic axes of Norway maple: from 50% at the first stage of desiccation to 150% in the next stages for APX, 50–150% for MR, 50–130% for DHAR and 50–170% for GR, and in sycamore: 50–150%, 20–80%, 20–80% and 20–70%, respectively. However, their activities expressed per mg of protein were much higher in Norway maple (Fig. 11). In cotyledons of seeds of this species, the activities of those enzymes did not increase during desiccation, but even slightly decreased in contrast to cotyledons of sycamore, where their activities slightly increased respectively: 0–50% for APX, 0–40% for MR, 0–30% for DHAR, and 0–20% for GR. Moreover in cotyledons of Norway maple dried to 13% MC, the MR activity completely disappeared. GPX activity during seed desiccation was nearly twice as



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high in Norway maple (10–100% in axes and 10–80% in cotyledons) than in sycamore (3–50% in axes and 10–40% in cotyledons) (Fig. 11). Native electrophoresis (SDS–PAGE) of APX confirmed that APX activity increased during desiccation for both Norway maple seeds and sycamore seeds (Fig. 12). However, GSHdependent DHAR activity was significantly higher for Norway maple seeds than for sycamore seeds (Fig. 13). Protein-bound thiol content declined significantly during desiccations in Norway maple seeds, but remained about the same or even increased slightly in sycamore seeds (Fig. 14). This was confirmed by electrophoretic separation of proteins from embryonic axes and cotyledons of studied seeds in three stages of desiccation, with detection of thiol groups

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Water content (%) Fig. 7. The germination capacity (open symbols) and electrolyte leakage (closed symbols) in seeds of A. platanoides ( , ) and A. pseudoplatanus (䊐, 䊏) during desiccation.

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3.0

O2–(OD530 g–1 DW)

Fig. 5. Changes in the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in embryonic axes and cotyledons of A. platanoides and A. pseudoplatanus seeds during development. Data are means of six replicates ± s.d. 1 unit = 1nmol ASA min−1 mg protein−1 (APX), 1 nmol NADH min−1 mg protein−1 (MR), 1 nmol ASA min−1 mg protein−1 (DHAR), 1 nmol NADPH min−1 mg protein−1 (GR).

0 60/53

**

**

2.5

H2O2–(nmol g–1 DW)

GR (units)

100

Electrolyte leakage (%)

600

Germination (%)

DHAR (units)

A. platanoides

Fig. 6. Native PAGE of ascorbate peroxidase isoenzymes in A. platanoides and A. pseudoplatanus seeds during early (11 and 14 WAF) and late (19 and 22 WAF) stages of development. Equal amounts (50 µg) of proteins were loaded in each lane.

50 **

**

**

40

30

40

2.0

30

1.5

60

50

40

30

20

10

20

60

50

20

10

Water content (%)

Fig. 8. Levels of superoxide oxygen radical and hydrogen peroxide in whole seeds of A. platanoides ( ) and A. pseudoplatanus (䊏) during desiccation. Data are means of three replicates ± s.d.

•

by the use of mBBr (Fig. 15). In embryonic axes, the level of thiol groups was significantly lower for Norway ample seeds than for sycamore seeds, even before desiccations.


ASA + DHA nmoles cotyledons–1

ASA + DHA nmoles axis–1

608


80 60

ASA Embryonic axes DHA

A. platanoides 0.7

2.3

1.5

6.0

2.1

5.8

53

43

36

26

13

7

A. pseudoplatanus

Embryonic axes

0.6

0.8

0.8

0.9

1.3

1.5

60

47

40

29

13

7

40 20 0

300

Cotyledons

A. platanoides 0.9

5.7

1.0

6.1

3.6

53

43

36

26

13

0.8

13.2

A. pseudoplatanus Cotyledons 2.1

0.9

1.4

1.7

4.1

47

40

29

13

7

200

100

0

7

60

Water content (%)

GSH + GSSG nmoles cotyledon–1 GSH + GSSG nmoles axis–1

Fig. 9. Changes in ascorbate (ASA) and dehydroascorbate (DHA) contents and ASA : DHA ratio in embryonic axes and cotyledons of A. platanoides and A. pseudoplatanus seeds during desiccation. Data are means of six replicates ± s.d.

15 12

Embryonic axes

A. platanoides 1.7 0.8

9

**

2.4 **

2.2 **

2.0 **

A. pseudoplatanus

Embryonic axes

0.6

6

0.3

0.4

1.1

0.4

1.0

0.4

60

47

40

29

13

7

3 0

180

52

36

26

A. platanoides

150

13

7

Cotyledons

A. pseudoplatanus

Cotyledons

4.0

120 90

43

0.8

**

60

2.1

2.0

**

**

36

26

1.7

1.1 0.6

1.2

1.6

2.0

0.6

0.6

60

47

40

29

13

7

30 0

52

43

13

7

Water content (%) Fig. 10. Changes in the contents of reduced (GSH) and oxidized (GSSG) forms of glutathione and GSH : GSSG ratio in embryonic axes and cotyledons of A. platanoides and A. pseudoplatanus seeds during desiccation. Data are means of six replicates ± s.d.

In cotyledons, the level of thiol groups decreased during desiccation for Norway maple seeds, and increased for sycamore seeds. Discussion There were significant differences in some components of the A–G system between orthodox and recalcitrant seeds of two

closely related Acer species. The study period encompassed embryogenesis (partly), seed filling, maturation and desiccation. Developmental changes were observed until seeds of both species were fully mature. However, based on the moisture content measured in the embryonic axes and cotyledons, desiccation at this time was not yet advanced in Norway maple seeds.



600

APX (units)

A. plat. A. pseud.

200

200

MR (units) DHAR (units)

100

0 52 60

43 47

36 40

26 29

13 13

1800

600

1500

500

1200

400

900

300

600

200

300

100

0

43 47

36 40

26 29

13 13 300

400

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200

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52 60

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13 13

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2000

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1500

2000

1000

1000

500

0

52 60

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0

13 13

50 40

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13 13

52 60

43 47

36 40

26 29

13 13

52 60

43 47

36 40

26 29

13 13

10 A. plat. A. pseud.

Embryaric axes

Cotyledons 8

30

6

20

4

10

2

0

52 60

0 52 60

600

0

GPX (units)

Cotyledons

300

400

0

GR (units)

Embryaric axes

52 60

43 47

36 40

26 29

13 13

0

52 60

43 47

36 40

26 29

13 13

Water content (%) Fig. 11. Changes in the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and glutathione peroxidase (GPX) in embryonic axes and cotyledons of A. platanoides and A. pseudoplatanus seeds during development. Data are means of six, replicates, ± s.d. 1 unit = 1 nmol ASA min−1 mg protein−1 (APX), 1 nmol NADH min−1 mg protein−1 (MR), 1 nmol ASA min−1 mg protein−1 (DHAR), 1 nmol NADPH min−1 mg protein−1 (GR), 1 nmol NADPH min−1 mg protein−1 (GPX).

The results show that orthodox seeds do not differ remarkably from recalcitrant seeds in ASA content nor do they differ in their redox status from the seed filling stage (18 and 15 WAF, respectively) until the end of seed maturation. Greater differences were observed at earlier stages of development, but this was probably because the rate of seed development

609

differed between species. Maximal values of the ASA : DHA ratio were recorded in Norway maple seeds at 14 WAF (1.7 for embryonic axes and 5.8 for cotyledons), whereas in sycamore seeds at 13 WAF (2.0 and 5.0, respectively). High values of ASA : DHA ratio are characteristic for periods of active cell divisions (Arrigoni et al. 1992). Also the high activities of APX and of the enzymes of ASA recycling (MR in particular) indicate that embryogenesis lasted until 13 WAF in seeds of sycamore. In embryonic axes, a marked decrease in MR activity was accompanied by a temporary increase in DHA level, which was observed at 16 WAF in Norway maple and at 15 WAF in sycamore. In cotyledons this symptom was less conspicuous. The two processes are correlated (Arrigoni 1994). Actively dividing cells use large amounts of ASA (Liso et al. 1984). Therefore, a high level of MR activity is needed in actively dividing cells (Paciolla et al. 2001; Potters et al. 2002). After the completion of embryogenesis, the activities of the enzymes of the A–G cycle were lower and stable. This was confirmed by the results of native PAGE of APX. At full maturity, there were no significant differences between the species in terms of ASA content and APX, MR and DHAR activity as there had been in previous studies on orthodox and recalcitrant seeds of other plant species (Tommasi et al. 1999). Very clear differences between the studied species were found in GSH levels and the GSH : GSSG ratio. A remarkable increase in GSH content, starting from 18 WAF in embryonic axes and cotyledons of seeds of Norway maple, was correlated with acquisition of desiccation tolerance. This indicates that GSH plays an important role in this process. In mature orthodox seeds, GSH level was nearly twice or three times as high as in recalcitrant seeds (in embryonic axes and cotyledons, respectively). Also the GSH : GSSG ratio throughout the period of seed maturation was much higher in orthodox seeds than in recalcitrant seeds (twice as high in embryonic axes and three times as high in cotyledons). The results suggest that orthodox seeds are prepared for water loss and, thus, for scavenging of the excess of ROS already before seed dehydration on the tree, owing to the high GSH content and high reducing intracellular redox environment, both in embryo axes and in cotyledons. Based on the data for ASA content, DHA content, and ASA : DHA ratio, it is difficult to determine what role the ascorbate system plays in preparing seeds for desiccation. There were no significant differences in ascorbic acid content and APX activity between the orthodox seeds and the recalcitrant seeds as there had been in other studies (Tommasi et al. 1999). Desiccation is a natural stage of seed development. Desiccation enables seeds to limit metabolism and survive for a long time under adverse conditions without losing their ability to germinate. This process is accompanied by increased ROS production, which is caused by perturbations of metabolic balance resulting from the partial dehydration of cells (Vertucci and Farrant 1995; Bailly 2004). The tolerance of orthodox seeds to desiccation is partly a result of the ability of cells to scavenge ROS in order to prevent their destructive activity (Leprince et al. 1993; Vertucci and Farrant 1995). In this study, there was an increase in the production of ROS during desiccation in both species. The increase was generally larger for sycamore maple seeds (Fig. 8). However, the site where ROS are

610



Acer platanoides (A)

Acer pseudoplatanus

(B )

53 43

36

26

13

(A)

53 43

36

26

13

60 47

(B )

40

29

13

60

47

40

29 13

Water content (%) Fig. 12. Native PAGE of ascorbate peroxidase in embryonic axes (A) and cotyledons (B) of A. platanoides and A. pseudoplatanus seeds at different stages of desiccation. Equal amounts (30 µg) of proteins were loaded in each lane.

Acer platanoides (B )

(A)

53

43

36

26

13

53 43

(A)

36

26

13

60 47

Acer pseudoplatanus (B )

40

29

13

60

47 40

29 13

Water content (%) Fig. 13. Native PAGE of DHAR in embryonic axes (A) and cotyledons (B) of A. platanoides and A. pseudoplatanus seeds at different stages of desiccation. Equal amounts (30 µg) of proteins were loaded in each lane.

produced and scavenged may determine the level of tolerance to desiccation more so than the total content of reactive oxygen species itself. Detoxification and the maintenance of cellular metabolic balance involve the antioxidant system, including A–G cycle. The ascorbate system does not play a role in post-maturation desiccation in many species with orthodox seeds, including Vicia faba, Avena sativa, Pinus pinea, Phaseolus vulgaris, Helianthus annuus and Triticum durum (Tommasi et al. 1999; Bailly et al. 2001, 2003; De Gara et al. 2003). This research showed that in some orthodox seeds the ascorbate system is ready until the end of seed maturation for defence against ROS during desiccation. This group includes the seeds of Norway maple as well as Fagus sylvatica (Pukacka and Wójkiewicz 2003). In this study APX activity before desiccation was about the same in Norway maple seeds and sycamore seeds and increased during seed desiccation (Figs 6, 12). Also the concentration of the substrate (ASA) for H2 O2 detoxification was similar in both types of seeds. However, the product of this reaction, DHA, was

more effectively reduced to the initial form in seeds of Norway maple than in seeds of sycamore. This is confirmed by the fact that DHAR activity was higher and DHA content was lower during desiccation in Norway maple seeds than in sycamore seeds (Figs 9, 11, 13). For this reason, the redox status of ASA in cells of orthodox seeds is markedly higher than in recalcitrant seeds (Fig. 9). This fact, together with an increased APX activity in embryo axes, suggests that the ascorbate system can play an important role in maintaining the metabolic balance in orthodox seeds during desiccation. For DHA reduction, the DHAR activity and presence of GSH and NADPH are necessary. The glutathione pool in orthodox seeds is several times larger than in recalcitrant seeds (Figs 4, 10). In orthodox seeds, no decrease in GSH content is observed during desiccation, and even a marked increase is observed at the initial phase of dehydration. This indicates that the defence system was mobilised in Norway maple seeds. Glutathione, apart from its participation in enzymatic H2 O2 detoxification by means of enzymes of the A–G cycle and



–SH groups (µg seed–1)

40 A. platanoides

57% 27% 12%

A. pseudoplatanus

60% 27% 13%

30

20

10

0

–SH groups (µg seed–1)

40

30

20

10

0

Non prot. –SH

Total –SH

Prot. –SH

Fig. 14. Levels of total, protein bound and non-protein thiols in A. platanoides and A. pseudoplatanus seeds during desiccation. Data are means of six replicates ± s.d.

GPX, can also indirectly react with ROS (Noctor and Foyer 1998). However, GSSG did not accumulate in the cells of the Norway maple seeds in this study. This suggests that in those seeds GSH synthesis must be increased and there

(A) 85 47

must be a very active system of removal of GSSG, which is harmful to cells (Schafer and Beuttner 2001; Kranner et al. 2006). Presented results showed that GR activity in Norway maple seeds is considerably higher than in sycamore seeds (Fig. 11). GSSG can also be bound to sulfhydryl groups on proteins and form of disulfide bridges, in a process called glutathionylation (Figs 14, 15) (Buchanan and Balmer 2005). This is favourable for orthodox seeds for two reasons: they increase protein stability and protect the seeds against the unfavourable effects of dehydration, and increase the redox status of glutathione in cells (Fig. 10). The high redox status of glutathione and ASA in cells of Norway maple seeds may be one of the main reasons why these seeds retain viability during desiccation (Kranner et al. 2006). Seeds of sycamore, because of a much smaller glutathione pool at the end of seed maturation and the lower activity of antioxidant enzymes, lose viability during desiccation. There is also a strong positive correlation between redox status of glutathione and seed viability during desiccation in Acer saccharinum seeds, which are more sensitive to desiccation than sycamore seeds (Pukacka and Ratajczak 2006). Seeds of the genus Acer have another adaptation to environmental conditions. In Norway maple seeds, moisture content can fall to 27%, even before shedding. In sycamore maple seed, moisture content does not fall below 40%, even in winter. This is because sycamore maple seeds have thick coats (S. Pukacka, unpubl. data). Sycamore seeds are protected from frost damage by the pubescent lining of the inner surface of the pericarp, and by the phenomenon of supercooling of freezable water (Pukacka and Pukacki 1997). Thus, seeds of sycamore, by means of avoiding dehydration and the development of some mechanisms enabling survival at low temperatures, have adapted to survive the unfavourable conditions in winter and to preserve the ability to germinate in their natural environment. In contrast, in seeds of Norway maple, an undoubtedly important element of cell protection against damage caused by dehydration is the A–G system and glutathione metabolism, which scavenge ROS and protect proteins against denaturation.

(B ) 1

2

3

4

5

611

6

1

2

3

4

5

6

85 47

36

36

26

26

20

20

kDa

kDa

Fig. 15. The presence of –SH groups, labelled with mBBr, in proteins extracted from embryonic axes (A) and cotyledons (B) of A. platanoides (1, 2, 3) and A. pseudoplatanus (4, 5, 6) seeds during desiccation. MC of seeds: Acer platanoides line 1–57%, line 2–27%, line 3–12%, Acer pseudoplatanus line 4–60%, line 5–27%, line 6–13%.

612


Acknowledgements This study was supported by research funds of the Polish Ministry of Sciences and Education.

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Manuscript received 20 January 2007, accepted 4 April 2007

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