Antioxidant defence and damage in senescing lupin nodules

Plant Physiol. Biochem. 40 (2002) 645–657 www.elsevier.com/locate/plaphy

Antioxidant defence and damage in senescing lupin nodules María Jesús Hernández-Jiménez, M. Mercedes Lucas, María Rosario de Felipe * Departamento de Fisiología y Bioquímica Vegetal, Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Científicas, Serrano, 115 Dpdo 28006-Madrid, Spain Received 7 December 2001; accepted 27 March 2002

Abstract Dark-induced and natural nodule senescence (ageing) were studied in lupin (Lupinus albus L. cv. Multolupa) plants. Continuous darkness caused loss of nitrogen fixation. After 2 d of treatment the nitrogenase (Nase, EC 1.18.6.1) activity decreased by 90%, and after 4 d it was completely abolished. Elevated catalytic iron content was detected in the nodule cytosol after 2–7 d of dark and after 7–9 weeks of nodule development versus mature 5-week-old nodules. Accumulation of ferritin, a protein involved in the storage of iron, occurred after 4–7 d of dark. The ascorbate content of nodules declined after 2–7 d of dark as well as in 8 weeks-old plants. The concentration of reduced thiols was lowered with darkness but augmented during ageing; however, the oxidized/reduced thiol ratio was enhanced in both circumstances. There were significant increases in the activity of superoxide dismutases (SOD, EC 1.15.1.1), glutathione reductase (GR, EC 1.6.4.2) and peroxidases in senescing nodules, and decreases in the catalase (EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities. The dehydroascorbate reductase (DHAR, EC 1.8.5.1.) activity also increased in dark stress but decreased during ageing. The content of leghaemoglobin decreased and protein carbonyl groups rose similarly at advanced stages of dark-induced and natural senescence. However, enhanced levels of malondialdehyde (MDA) only were detected during ageing. Overall, these data support the idea that nodule senescence is related to an increase in oxidative stress. Concerning the ultrastructural alterations, a strong degree of similarity has been found between dark-induced and natural senescence. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Antioxidant defence; Darkness; Ferritin; Iron; Lupinus; Nodule; Senescence

1. Introduction In legume root nodules, nitrogen fixation is particularly sensitive to oxygen and reactive oxygen species (ROS) [28]. The formation of superoxide (O2–•), hydrogen peroxide (H2O2) and hydroxyl radical (OH•) is highly potential in nodules because of the strong rate of respiration and the

Abbreviations: APX, ascorbate peroxidase; ASC, ascorbate; DHAR, dehydroascorbate reductase; EDTA, ethylenediaminetetraacetic acid; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; HPLC/UV, high performance liquid chromatography/ultraviolet; H2O2, hydrogen peroxide; Lb, leghaemoglobin; MDA, malondialdehyde; MDHAR, monodehydroascorbate reductase; mRNA, messenger ribonucleic acids; OH•, hydroxyl radical; O2–•, superoxide anion; PVP, polyvinylpirrolidone; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SOD, superoxide dismutase * Corresponding author. E-mail address: [email protected] (M.R. de Felipe). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 2 2 - 5

high concentration of leghaemoglobin (1–3 mM). The oxygenated form of Lb is subjected to autoxidation with release of superoxide that disproportionates to H2O2 [27]. On the other hand, the very active iron metabolism in the nodule seems to be closely related to free radical reactions and oxidative stress [4]. Free iron catalyses the Fenton reaction (H2O2 + Fe2+ → Fe3+ + OH• + OH–), resulting in the production of the highly reactive OH• radical, which can attack swiftly almost every molecule at its formation site. Hydroxyl radicals can damage sugars, lipids, proteins and DNA. In addition, free iron facilitates the decomposition of lipid hydroperoxides with the subsequent generation of the harmful alkoxyl and peroxyl radicals. Moreover, iron is also involved in the generation of O2–• and H2O2 by accelerating the non-enzymatic oxidation of several molecules as glutathione. Hence it can be argued that ferritin, an iron-storage protein, is an antioxidant mechanism that ensures the absence of Fe ions by metal sequestration [16].

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Additionally, antioxidant defence is largely provided by specific enzymes and small molecules. These include the superoxide dismutases (SOD) that detoxify O2–•, and peroxidases, catalases and the enzymes of the ascorbate–glutathione (ASC–GSH) cycle associated with the scavenging of H2O2 [3]. Nodules are also equipped with ascorbate and glutathione that can act directly as antioxidant by removing H2O2 and O2–•. The balance between ROS formation and their removal safeguards cells from oxidative damage. In soybean nodules, the occurrence of oxidative stress, apparently due to the enhanced formation of ROS, has been proposed to be involved in natural senescence [6]. Besides enhanced production of oxidants, reduction of antioxidant defences has been reported to occur during stress-induced senescence [9,18]. The exposure of nodulated legume plants to darkness has been used as a model system to induce premature senescence in nodules [9,32], and it has been suggested that the mechanisms underlying dark-induced and natural senescence are related [36]. Nevertheless, the comprehensive comparison of the effects of prolonged dark and ageing on the mechanisms of antioxidant protection in nodules is not well documented. In addition, the precise sequence of the biochemical changes leading to loss of function and structural deterioration of nodules is far from clear. Stress tolerance and senescence rates appear to depend on the legume species, and these differences may be related to some extent to the growth pattern of nodules [30]. The goal of the present study was to compare the mechanisms involved in dark-induced and natural senescence in lupin root (indeterminate) nodules. Parameters examined include ROS detoxification systems, catalytic iron content, oxidative damage to biomolecules and ultrastructural alterations.

2. Results 2.1. Effect of darkness on nitrogenase activity and changes in protein content of senescent nodules The darkness drastically inhibited the nitrogenase activity. After 2 d of stress, the activity per plant, as well as per nodule fresh weight, decreased a 90%, and after 4 d it was completely abolished. The protein content in 5-week-old nodules was approximately 20 mg g–1 of fresh weight. The protein concentration decreased about 25 and 45% following 6 and 7 d of dark, respectively. Similarly, a decline of 55% was observed in nodules of 9- versus 5-week-old plants. 2.2. Changes in protein SDS-PAGE pattern, leghaemoglobin and ferritin content Regarding the electrophoretic pattern of soluble proteins, several changes were observed after 7 d of darkness: en-

Fig. 1. SDS-PAGE electrophoresis of total cytosolic proteins and Lb immunoblot in lupin nodules. A, dark treatments. Lanes 1, 7-d-dark small nodules; 2, 7-d-control small nodules; 3, 7-d-control large nodules; 4, 7-d-dark large nodules. B, natural senescence: Lanes 1, 5-week-old nodules; 2, 7-week-old nodules; 3, 9-week-old nodules. Arrows on the left-hand side indicate variations of polypeptides after dark treatments and ageing. Protein molecular mass markers are aligned on the right-hand side.

hanced level of the polypeptides corresponding to 28, 26, 24, 19 and 16 kDa, presence of a new polypeptide of 39 kDa, and decline of leghaemoglobin content, as confirmed by western analysis (Fig. 1A). To test whether differences in the ferritin content occur during dark-induced senescence in nodules, immunoblot analysis was carried out using a polyclonal antibody raised

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Table 1 Content of catalytic iron, carbonyl groups and malondialdehyde (MDA) in senescing nodules treated with darkness for 0, 2, 4, 5, 6 and 7 d. For each parameter, means (n = 5–9) denoted by the same letter do not differ sigificantly at p < 0.05 according to Ducans’s multiple range test Parameter (nmol mg–1 protein)

Days 0

Catalytic iron Carbonyl groups Malondialdehyde

Fig. 2. Ferritin immunoblot of nodules extracted with 100 mM (pH 6.5) potassium phosphate buffer and 75 min of blotting. Lanes 1, 4-d-control large nodules; 2, 4-d-dark large nodules; 3, 7-d-control large nodules; 4, 7-d-dark large nodules. Arrows on the left-hand side indicate poypeptides of 28, 26 and 24 kDa. Protein molecular mass markers are aligned on the right-hand side.

2

0.57 8.9 a 1.2 a

a

4 b

0.79 9.4 a 0.68 b

5

6

b

c

0.77 0.95 11.6 a 8.3 a 0.60b 0.58 b

7 d

1.51 2.29e 12.5 b 16.2 c 0.85 c 1.1 a

ide, prior to the staining procedure, showed that SOD-1 and -2 were Mn-SODs, and SOD-3, -4 and -5 corresponded to Cu/Zn-SODs (Fig. 3B). Increased staining of SOD-1 and SOD-4 were observed after 5–7 d of dark (Fig. 3A). The total SOD activity was also elevated during ageing (Table 4). A rise of 45% in the total activity occurred in 8-week-old nodules, and an increase above 100% was found in 9- versus 5-week-old nodules (Table 4), resulting from the enhanced activity of the isoenzyme SOD-1, -2 and -3 (Fig. 3B).

against pea seed ferritin. Interestingly, the anti-ferritin serum reacted with three polypeptides of 28, 26 and 24 kDa. In control nodules, the band of 26 kDa was the most intensely immunolabelled but the band of 24 kDa was faintly perceptible (Fig. 2). An increase of the polypeptide of 24 kDa, not discernible by Coomassie blue staining, was detected after 4 d of dark. No further significant changes were observed until 7 d of dark. Then, increased level of the polypeptide of 26 kDa and an intense augmentation in the immunostaining of the polypeptide of 24 kDa were found (Fig. 2). Modifications in the SDS-PAGE profile of proteins appeared evident in 9-week-old nodules: increased amounts of the polypeptides of 28, 26, 24 and 16 kDa, diminished level of a polypeptide of molecular mass higher than 92 kDa, significant decrease of the polypeptides of 78 and 33 kDa, and decreased leghemoglobin (Lb) content (Fig. 1B).

2.4.2. Enzymes involved in the removal of hydrogen peroxide To investigate the changes in the scavenging of H2O2, the activity of catalases, peroxidases and the enzymes of the ASC–GSH cycle were analysed. The visualization of catalase activity on gels showed the existence of three isoenzymes in lupin nodules. Following 7 d of dark, the activity of isoenzymes 1 and 2 was lowered whereas the activity of isoenzyme 3 was slightly increased (Fig. 4). During ageing, a decrease in the activity of the three isoenzymes was perceived in 9-week-old nodules (Fig. 4). Total nodule

2.3. Variations of catalytic iron content

Parameter (nmol mg–1 protein)

Prolonged darkness and ageing enhanced the catalytic Fe content in the cytosolic fraction of nodules (Tables 1 and 2). After 2 d of darkness, catalytic Fe concentration increased by 38%, and multiplied by four after 7 d of stress (Table 1). Likewise, a rise of 66% was observed in nodules of 7versus 5-week-old plants, and further increase up to 4.5 times occurred in 9-week-old plants (Table 2). 2.4. Variations of antioxidant enzymes activities 2.4.1. Superoxide dismutases Darkness induced a moderate increase (34%) in the total SOD activity in nodules after 5 d, and an enhancement of 63% occurred after 7 d (Table 3). The activity of specific isoenzymes was visualized on gels. The staining of SODs revealed the presence of five isoenzymes (Fig. 3). The incubation of gels in potassium cyanide or hydrogen perox-

Table 2 Content of catalytic iron, carbonyl groups and malondialdehyde (MDA) during nodule ageing. Statistical analysis of means (n = 6–9) was performed as for Table 1 Weeks 5

7

0.6 a 8.8 a 1.2 a

Catalytic iron Carbonyl groups Malondialdehyde

8

1.0b 10.2 a 1.2 a

9

1.0b 11.1 a 1.3 a

2.7 c 15.0b 2.8b

Table 3 Enzyme activities of SOD and the ascorbate–glutathione cycle in senescing nodules treated with darkness for 0, 4, 5, 6 and 7 d. Data are expressed in nkatals g–1 protein except for SOD. *units SOD mg–1 protein as described in Methods. Statistical analysis of means (n = 6) was performed as for Table 1 Enzyme

Days 0

4 a

5 b

6 b

7 b

12.0 14.0 13.8 17.0 c Superoxide dismutase* 10.4 Ascorbate peroxidase 5016 a 4980 a 4416 a 4516 a 4130 b DHA reductase 5.7 a 6.0 a 5.7 a 7.2 b 10.7 c MDHA reductase 230 a 198 a 192 a 210 a 120 b Glutathione reductase 393 a 598 b 857 c 843 c 1060 d

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Fig. 4. Effect of natural and dark-induced senescence on catalase isoenzymes of lupin nodules: Lanes 1, 9-week-old nodules; 2, 5-week-old nodules (= 7-d-control nodules,); 3, 7-d-dark nodules.

Fig. 3. Effect of darkness and natural senescence on SOD isoenzyme pattern of lupin nodules. A, dark treatments. Lanes 1, 5-d-control nodules; 2, 5-d-dark nodules; 3, 6-d-control nodules; 4, 6-d-dark nodules; 5, 7-d-control nodules; 6, 7-d-dark nodules. B, natural senescence. Lanes 1, 5-week-old nodules; 2, 8-week-old nodules; 3, 9-week-old nodules. KCN, gel incubated in potassium cyanide, 5-week-old nodules. H2O2, gel incubated with 5 mM hydrogen peroxide, 5-week-old nodules.

catalase activity decreased (35%) only when plants were exposed to continuous darkness for 7 d. Nodule ageing had a significant effect on catalase activity, which decreased ranging from 42% for 7-week-old plants to 88% for 9-week-old plants (data not shown). In contrast, dark enhanced the total peroxidase activity in the nodules after 4–7 d of treatment, and increasing activity was also detected during the ageing of nodules after 7 weeks (Fig. 5). Nevertheless, the ascorbate peroxidase activity was not increased in dark treatments, and even it was slightly reduced (18%) following 7 d of stress (Table 3). During natural senescence, a diminution of 50% occurred in the 9versus the 5-week-old nodules (Table 4).

Concerning to the other enzymes of the ASC–GSH cycle, several changes were found (Tables 3 and 4). Following 4 d of dark, the GR activity was increased about a 50%, and more prominent rise was found after 5–7 d. Dark increased the DHAR activity (26% after 6 d and 88% after 7 d), but lowered by 50% the MDHAR activity after 7 d of treatment. Similarly, GR activity augmented during ageing (25% in 8-week-old and 96% in 9-week-old nodules), but the DHAR and MDHAR activities decreased (45 and 20%, respectively) in the 9-week-old nodules. 2.5. Variations of antioxidant metabolites 2.5.1. Glutathione Considering that the method used for measuring the content of glutathione does not distinguish it from other γ-glutamyl-cysteinyl-tripeptides found in leguminous plants, the data that we report in this work may correspond to the total of glutathione plus other analogous tripeptide

Table 4 Enzyme activities of SOD and the ascorbate–glutathione cycle during nodule ageing. Data are expressed in nkatals g–1 protein except for SOD. * units SOD mg–1 protein as described in Methods. Statistical analysis of means (n = 6) was performed as for Table 1 Enzyme

Weeks 5

Superoxide dismutase* Ascorbate peroxidase DHA reductase MDHA reductase Glutathione reductase

7 a

10.5 4966 a 5.2 a 233 a 433 a

8 a

10.7 4083 a 6.3 a 205 a 450 a

9 b

15.2 4133 a 4.8 a 248 a 535 b

21.5 c 2566 b 2.8 b 185 b 856 c

Fig. 5. Effect of darkness and ageing on peroxidase enzyme of Lupinus albus. The pseudo-peroxidase activity of Lb components (Lb I and Lb II) is also shown. Lanes 1, 4-d-control nodules; 2, 4-d-dark nodules; 3, 7-d-control nodules nodules; 4, 7-d-dark nodules; 5, 5-week-old nodules; 6, 7-week-old nodules; 7, 9-week-old nodules.

M.J. Hernández-Jiménez et al. / Plant Physiol. Biochem. 40 (2002) 645–657 Table 5 Content of ascorbate, reduced and oxidized thiols (glutathione) in senescing nodules treated with darkness for 0, 2, 4, 5, 6 and 7 d. Statistical analysis of means (n = 5–9) was performed as for Table 1 Days

Metabolite (nmol g–1 fresh weight) 0

2

4

5

6

7

Ascorbate 139 a 90 b 70 c 18 d 13 d 11 d Reduced 1130 a 1124 a 1118 a 395b 400 b 410 b thiols (GSH) Oxidized 108 a 112 a 115 b 60 b 58 b 74 a thiols (GSSG) GSSG/GSH 0.09 a 0.10 a 0.10 a 0.15 b 0.15 b 0.18 c ratio

derivates possibly present in lupin nodules. Nevertheless, since the role of GSH in antioxidant defence is related to the cysteine moiety of the tripeptide, the homologues might exercise a similar function in the scavenging of ROS. Therefore, we consider that the interpretation of the following results is still valid in terms of antioxidant protection of nodules. Prolonged dark treatments substantially lowered the content of GSH in the nodules by approximately 65% after 5 d, whereas the GSSG decreased about 40%, leading to an increased GSSG/GSH ratio (Table 5). In contrast, during natural senescence the response of glutathione content was distinct. Reduced and oxidized forms of glutathione rose significantly in the nodules of 9-week-old plants, and the GSSG/GSH ratio also increased (Table 4). 2.5.2. Ascorbate The total content of ascorbate decreased after 2 d of dark (35%), and it was 92% lower after 7 d of darkness (Table 5). With ageing, the total ascorbate content decreased by 65% in 8-week-old nodules and by 90% in 9-week-old nodules (Table 6). 2.6. Oxidative damage to biomolecules: proteins and lipids Dark stress did not change the oxidized protein content, estimated as carbonyl groups, after 5 d of dark, but caused increases of 40 and 80% after 6 and 7 d of treatment, respectively (Table 1). During ageing, protein oxidation was Table 6 Content of ascorbate, reduced and oxidized thiols (glutathione) during nodule ageing. Statistical analysis of means (n = 6–9) was performed as for Table 1 Metabolite (nmol g–1 fresh weight)

enhanced about a 70% in 9-week-old nodules versus 5-week-old nodules (Table 2). MDA content was 50% lower in nodules after 2, 4, and 5 d of dark, whereas only decreased by 30% at 6 d, and no significant difference was found after 7 d of dark versus the control (Table 1). In contrast, MDA level increased in senescing nodules of 9-week-old plants versus mature 5-week-old nodules (Table 2). 2.7. Ultrastructural alterations To complement the biochemical results, nodule ultrastructure was examined during dark-induced and natural senescence by electron microscopy. 2.7.1. Dark-induced senescence In the infected zone of mature nodules, the cells were densely packed with symbiosomes, enclosing a single bacteroid (Fig. 6A). Two days of dark only affected a limited number of infected cells in the interior of the nodules (Fig. 6B). Following 4 d of stress, the bacteroids showed disrupted cytosol and the host cell cytosol appeared highly dense to the electrons in some cells (Fig. 6C). As senescence progressed, cytosol breakdown and organelle changes were observed (Fig. 6D). Interestingly, a few unaltered cells were also found in the senescing zone. Furthermore, unaltered bacteria were sporadically found in the intercellular spaces, together with membrane structures that probably resulted from bacteria degeneration (Fig. 6B). The senescing zone extended during longer dark treatment. After 7 d of dark, the host cell cytosol was completely degraded and breaks in the symbiosome membrane were discernible (Fig. 7). Intriguingly, we observed large intercellular spaces filled with unaltered bacteria, which were not enclosed by membranes (Fig. 7). 2.7.2. Natural senescence The ultrastructural study of natural senescence was carried out from 5 to 12 weeks after sowing (Fig. 8). The first symptoms of senescence in the infected cells were found in 7-week-old nodules (Fig. 8B). The pattern of ultrastructural alterations resembled that described above for dark stress. Similarly, the symbiosome membrane was only disrupted when severe symptoms of degeneration became widespread (Fig. 8D). In the infected cells of 12-week-old nodules, the cytosol breakdown was drastic and scattered membrane structures were often observed (Fig. 8E). Despite the evident degeneration of these cells, the amyloplasts did not show visible alterations (Fig. 8E).

Weeks

3. Discussion 5

Ascorbate Reduced thiols (GSH) Oxidized thiols (GSSG) GSSG/GSH ratio

649

7

8

9

138 a 51 b 13 c 140 a 1120 a 1128 a 1114 a 4100 b 110 a 114 a 106 a 670 b 0.10 a 0.10 a 0.09 a 0.16 b

This study shows that natural and dark-senescence are not identical but very similar in lupin nodules. Common characteristics include protein modifications, several responses of ROS systems and ultrastructural alterations.

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Fig. 6. Electron micrographs showing the effect of 4 d darkness on lupin nodules. A, control infected cells; B, infected cells showing altered symbiosomes and non-affected bacteria in the intercellular spaces; C, infected cells showing altered cytosol and symbiosomes; D, infected cells very deteriorated, showing the disruption of cell organelles; E, alterations in nuclei of infected cells. AM, amyloplasts; B, bacteroids; BM, bacteroidal membrane; C, cytosol; CW, cell wall; D, DNA; G, Golgi apparatus, IE, intercellular spaces; M, mitochondria; N, nucleus; P, plastid; PBM, peribacteroidal membrane. Bar = 0.5 µm.

3.1. Catalytic iron, ferritin and leghaemoglobin in nodule senescence It is well known from many studies that nitrogen fixation in legume nodules is decreased under photosynthetic stress

induced by darkness, defoliation and other stresses [11,32]. The decrease of nitrogenase activity induced by nitrate in lupin nodules has been attributed to the decrease in Lb [35]. In contrast, our results show that N2 fixation declined after 2 d of dark whereas Lb content decreased only after 7 d. It

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Fig. 7. Electron micrograph showing the effect of 7 d darkness on infected cells. The symbiosomes are very much affected and also the cell cytosol. The intercellular spaces, however, show non-affected free-living bacteria. The abbreviations are as in Fig. 6. Bar = 0.5 µm.

is worth noting that an elevated catalytic Fe content was found after 2 d of dark. Similarly, the increased catalytic Fe content detected in 7-week-old nodules also preceded the diminution of Lb level during natural senescence of nodules. Since the total protein content remained stable after 2–5 d of dark and until 9 weeks of nodule development, changes in the mechanisms involved in Fe sequestration and mobilization probably would be the main cause of the augmentation of catalytic Fe concentration, rather than Fe release from degraded Lb and other haemoproteins. Regarding Fe storage, increases of ferritin content occurred in lupin nodules after 4 d of dark. Although the extent of iron loading cannot be estimated, ferritin accumulation may be interpreted as an antioxidant response. During natural senescence, we previously presented evidence of ferritin accumulation in the plastids and amyloplasts of cortex cells, but a decrease in the infected cells of senescing nodules in soybean and lupin plants as well as in the senescent zone of alfalfa nodules [16]. In lupin nodule extracts three polypeptides of 28, 26 and 24 kDa were immunodetected. In several plant species, the 26 kDa subunit has been supposed to be generated from the 28 kDa subunit by cleavage of the N-terminal region [1]. In the embryo axis of Pisum sativum a disappearance of ferritin was observed, which was accompanied by the appearance of ferritin fragments of lower molecular mass (26.5 and 25 kDa) than the basic 28 kDa subunit. These smaller polypeptides had the same molecular mass as those observed in vitro in response to free-radical damage produced by reduction-mediated iron release. A specific free-

radical cleavage at the N-terminal part of the 28 kDa subunit is likely to be the initial signal for ferritin degradation. Nevertheless, Masuda et al. [17] recently found that the 28 and 26.5 kDa subunits purified from dried soybean seeds had different amino acid sequences. Moreover, the two different ferritin subunits show differential sensitivity to protease digestions and the uncleaved 28 kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5 kDa subunit. Interestingly, the abundance of the 28 kDa ferritin subunit remained unchanged whereas increases in the abundance of 24 and 26 kDa subunits were observed in lupin nodules during dark-induced senescence. We previously showed that ferritin content considerably increased with nodule age in lupin and it was only related to the 28 and 26 kDa subunits (the 24 kDa subunit was almost imperceptible) [16]. It has been described that ferritin is posttranscriptionally regulated during nodule development in soybean, and the changes observed in nodule ferritin protein content could be due to increased ferritin turnover or autocatalytic degradation in mature nodules [15]. Whether similar regulation takes place during stress-induced nodule senescence should be investigated. In the present report, our results suggest that ferritin synthesis may be induced by an increased level of catalytic Fe in nodules during both dark-induced and natural senescence. This assumption is in good agreement with the recent report of iron-mediated derepression of the ferritin gene in soybean [37]. Furthermore, Briat et al. [1] have reported that H2O2 induces ferritin mRNA accumulation in derooted maize plantlets in the presence of low iron concentrations.

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Fig. 8. Electron micrographs showing the effect of ageing on infected cells. A, 5-week-old nodules with normal symbiosomes and cell cytosol; B, non-affected free living bacteria in the intercellular spaces in a 9-week-old nodule; C, 9-week-old infected cells showing very deteriorated symbiosomes and electron dense cytosol; D, 9-week-old infected cells showing peribacteroidal membranes and cytosol vesicles; E, 12-week-old nodule showing organelles and cytosol disruption. Amyloplasts are still visible. Arrows show rests of cellular membranes. BA, bacteria. The rest of the legend is as in Fig. 6. Bar = 0.5 µm.

Interestingly, this effect is prevented by pretreatment of plantlets with antioxidants, indicating that the induction of ferritin gene expression in this system requires an oxidative stress. In this respect, the decrease of ASC occurring during nodule senescence may be related in some way with the

increased amounts of ferritin proteins. In as much as Fe homeostasis is critical to prevent the formation of the harmful hydroxyl radical, further work is needed to ascertain the identity of each ferritin subunit, its functional role, and its regulation during nodule ageing and environmental stresses.

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3.2. Antioxidant defence during nodule senescence Exposure of lupin plants to 2 d of continuous dark reduced the total ascorbate content (ascorbate + dehydroascorbate) in nodules. This effect may be due to nonenzymatic oxidation of ascorbate, facilitated by an increased level of catalytic iron. Although no other significant differences were detected in antioxidant protection, a decrease in the ascorbate level may reduce the removal of hydrogen peroxide. This diffusible oxidant molecule can oxidize the ferrous form of Lb (active O2 transporter) and the Lb-ferric form to the inactive form of ferryl-Lb [4]. Ascorbate and glutathione can reduce the ferryl-Lb to the active ferrous Lb [26]. Since the oxyLb undergoes slow autoxidation to the ferric form [27], a decrease in ASC would weaken Lb protection. Consequently, oxygen transport to the bacteroid could be affected, resulting in a reduced nitrogenase activity. In these circumstances, the role of GSH in the protection of Lb would be particularly essential after 2 d of dark stress and after 8 weeks of nodule development. It is interesting to notice that increased GR activity was found and GSSG/GSH ratio remained stable after 4 d of dark treatment. Additionally, the total peroxidase activity was also elevated. Peroxidase activity might be expected to reduce the level of ROS by metabolizing H2O2, but peroxidase is also capable of various “oxidase” reactions leading to H2O2 generation. Furthermore, it has been shown that some plant peroxidases have a thiol oxidase function [24]. Decreases in the total ascorbate and glutathione content as well as the increase of the GSSG/GSH ratio may arise from and/or lead to elevated level of H2O2. Following 5 d of dark, the increase of SOD activity would modify the O2–•/H2O2 ratio and therefore could reduce the risk of formation of hydroxyl radicals by Fenton reactions. After 7 d of dark, the strong rise in SOD activity, together with the reduced catalase and MDHAR activities might elevate the level of H2O2. The increased DHAR activity would facilitate the rapid regeneration of the low amount of ascorbate. In spite of the enhanced GR activity, an important decrease of glutathione and increase of GSSG/GSH ratio occurred in the nodules of 7 d dark-stressed plants. An increase of oxidized thiols and decreases of GSH and hGSH have been found in pea nodules after 4 d of dark [18]. During lupin ageing, the SOD and GR activities also augmented in 9-week-old nodules. However, the activities of the other enzymes of the ASC–GSH cycle were negatively affected, decreasing the ability of elimination of H2O2 and of regeneration of reduced ascorbate. The strong diminution of ascorbate may lead to a rise of ROS and eventually to the development of oxidative stress. Total glutathione accumulated in 9-week-old nodules. However, the increased GSSG/GSH ratio indicates that the enhanced glutathione content was not sufficient by itself to buffer the high rate of AOS production. The oxidated/reduced ratio of the thiol compounds gluthatione and homogluthatione increased in soybean nodules during the overall period of

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nodule development but in all cases the percentage of the oxidized form remained low [6]. On the other hand, an increase of the glutathione pool and the DHAR activity was found in peroxisomes and soluble fractions of senescent pea leaves [13]. Our results and those cited above support the importance of glutathione regeneration in the protection against oxidative stress conditions during senescence. In lupin nodules the glutathione pool size is distinctly affected by darkness and ageing. The different stress conditions during these senescence processes may involve distinct mechanisms and, consequently, could evoke disparate responses. Since glutathione is involved in the regulation of gene expression and signal transduction [39], adjustment of the total concentration and ratio of the reduced and oxidized forms of this antioxidant may also be of regulatory significance. To date, the biochemical mechanisms through which glutathione content is modulated in nodules have not been well characterized. Candidates are altered rates of synthesis, degradation, import or export. To determine the aspects of thiol transport, as well as the regulation of glutathione synthesis and catabolism during senescence future experiments are required. Another different response observed during dark-induced and natural senescence was the alteration of isoformspecific catalase activity. Although a clear reduction of CAT-1 and CAT-2 activities was observed in both natural and induced senescence, a slightly increased CAT-3 activity was observed during ageing, which was not enough to overcome the decreased activities of CAT-1 and CAT-2 isoenzymes. To our knowledge there is no information about the catalase isoenzymes present in another legume nodules. The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form six isoenzymes in flowers and leaves and two isozymes in roots [20]. Recently, three isoformes of catalase in wheat peroxisomes, which responded differentially during monocarpic senescence [31]. On the other hand, it has been shown that the expression of two catalase genes in Arabidopsis is differentially controlled by the circardian clock [20]. Both, decreases and increases of the total catalase activity in dark-induced senescence in nodules have been reported (this work, [9,18]). Whether the different results obtained from different plants are related to either the type of nodule (indeterminate, determinate) or a differential regulation should be determined. 3.3. Oxidative damage to biomolecules The oxidative damage to proteins occurred in the advanced stages of senescence during ageing and dark stress in lupin nodules, indicating that increases in the formation of ROS are related to the loss of function of senescing nodules. These results are in agreement with those previously reported in dark-stressed common bean and pea nodules, although higher tolerance to this stress is found in lupin plants [9,18]. The high catalytic Fe content at ad-

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vanced stages of senescence would facilitate the production of OH•, and in consequence, the oxidation of proteins. The enhanced levels of oxidized proteins also correlated with the decline of total protein and Lb. It has been reported that protein synthesis in vitro is inhibited by GSSG [12]. Thus increases of GSSG/GSH ratio may reduce protein synthesis and promote protein oxidation and subsequent proteolysis. It is remarkable that SOD and GR activities increased in lupin nodules, suggesting that these enzymes are less susceptible to oxidation and proteolysis. It is also possible that the stability of their mRNAs plays an important role in the regulation of the levels of these enzymes. We suggest that the ability of lupin cells to induce SOD and GR activities is crucial in the delay of oxidative damage, and it explains at least partially their high tolerance to darkness. Lipid peroxidation, estimated as MDA content, increased during ageing in senescing lupin nodules. However, darkness decreased MDA levels after 2–5 d. In common bean plants, decreases of MDA content have also been reported in nodules exposed to nitrate [19] or dark stress [9], whereas enhanced levels have been found in nodules subjected to water stress [10]. It is well known that a huge diversity of products can be generated by lipid peroxidation in biological systems, including peroxyl radicals, hydrocarbon gases and aldehydes such as MDA. Since the thiobarbituric acid test used in this work measures MDA only, we cannot report accurate evidence of lipid damage in dark-induced senescence in lupin nodules.

3.4. Ultrastructure alterations during nodule senescence The effects of darkness on the structure of lupin nodules were very similar to the alterations observed during natural senescence. The initial ultrastructure modifications occurred concomitant with catalytic Fe content increase. The fusion of symbiosomes resulted in the formation of larger symbiosomes containing several bacteroids. Similar changes have been described during ageing of bean nodules, coinciding with the decline of nitrogenase activity [25]. In lupin, the degeneration of bacteroids took place inside the symbiosome and the breakdown of the symbiosome membrane (peribacteroid membrane) was also observed at very advanced stages of senescence. In contrast, the disruption of the symbiosome membrane occurs before the bacteroid disintegration in bean, soybean and alfalfa nodules [25]. Thereby, a protective role against the host proteases has been ascribed to the symbiosome membrane. On the contrary, Kijne [14] found degraded bacteroids inside large vesicules in the senescent zone of pea nodules. These structures remarkably resemble those observed in lupin. Furthermore, some authors have suggested the implication of bacteroid autolysis, although the role of heterophagic processes in bacteroid degeneration cannot be excluded [21].

Another interesting finding during lupin nodule senescence was the existence of a large number of bacteria in large intercellular spaces at advanced stages of senescence. Likewise, the herbicide linuron induces this effect in lupin nodules [7], suggesting that it is an adaptive response to physiological and induced stresses. In addition, some authors have proposed the saprophytic multiplication of bacteria by utilizing the products of nodule autolysis [34]. Moreover, recent results obtained with Lotus japonicus colonized by Rhizobium sp. NGR234 suggest a surprising high potential of redifferentiation of bacteroids into freeliving bacteria in senescing nodules [23]. Thus the results presented in this work are in agreement with current evolutionary models suggesting that nitrogen-fixing rhizobia in nodules (bacteroids) should have a higher survival probability, after nodule senescence, than free-living rhizobia in the rhizosphere. The presence of a large number of bacteria in the intercellular spaces of senescing lupin nodules deserves further investigation since it could provide some insights about how the rhizobia get out of nodules, and possibly how they infect the host cells in lupin roots. Since no infection threads have been observed in lupin nodules, the infection might occur through the intercellular spaces. Regarding the host cell, as it was previously showed in natural senescence [16], plastids and amyloplasts are the last organelles being destroyed during dark-induced senescence. Besides the existence of ferritin in these organelles, the specific localization of γ-glutamylcysteine synthetase [22], GR [33] and Fe-SOD [29], and the regulation of those enzymes by ROS support the hypothesis that those organelles play an essential role in the protection against active oxygen.

4. Methods 4.1. Plant growth, darkness treatments and measurement of nitrogen fixation Seeds of Lupinus albus L. cv. Multolupa were sterilized and inoculated with a suspension of Bradyrhizobium sp. Lupinus strain ISLU16 at the time of planting. Plants were grown in a growth chamber at 26 °C/16 h day and 20 °C/8 h night photoperiod and irrigated with nitrogen-free Hogland’s nutrient solution. After 30 d of planting, plants were placed in total darkness for 2, 4, 5, 6 and 7 d, then root nodules were harvested, cleaned with ice-cold distilled water and maintained at 2 or –20 °C until extracted. For natural senescence studies, plants were kept in the growth chamber at conditions mentioned above and harvested after 5, 7, 8 and 9 weeks after sowing and inoculation. Note that control plants of 7 d in dark are 5 weeks old, and we used nodules from these plants for both natural and dark-induced senescence experiments. Since nodules at different developing stages are present in the roots of aged plants, the N2 fixation rate was not considered as a precise marker of

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natural senescence. To study the time course of events leading to natural senescence, the largest nodules in the upper part of the roots, which also were the oldest ones, were carefully selected at each time point. Nitrogenase activity was measured by the acetylene reduction assay [7]. 4.2. Microscopy studies Samples were processed for light and electron microscopy using Araldite [16]. 4.3. Protein extraction, gel electrophoresis and immunoblotting The nodules were ground in 100 mM potassium phosphate buffer (pH 6.5), and the extract was processed as in Lucas et al. [16]. Protein content determination, fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, electroblotting and immunolabelling of ferritin and leghemoglobin were performed as described by Lucas et al. [16]. Polyacrylamide gels were stained with 0.25% (w/v) Coomassie Brilliant Blue in 50% (v/v) methanol and 10% (v/v) glacial acetic acid in water. 4.4. Enzyme assays Total SOD activity was analysed in total extracts of nodules as in De Lorenzo et al. [5] and one unit of activity was defined as the amount of enzyme that inhibits the rate of cyt c reduction by 50% at 25 °C. The activities of different isoenzymes were visualized on 10% polyacrylamide gel according to Weisiger and Fridovich [38]. Activity of catalase isoenzymes was assayed according to Woodbury et al. [40] in crude extracts of nodules, and total peroxidase activity was determined using o-dianisidine as a nonphysiological electron donor as described in De Lorenzo et al. [5]. The APX was extracted at 4 °C from 0.25 g of nodules in 1 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM ascorbate and 0.5% (w/v) PVP, with a mortar and pestle. The homogenate was centrifuged at 15 000 × g for 20 min at 4 °C. The APX activity was measured in the supernatant as described in Jiménez et al. [13]. DHAR, MDHAR and GR were extracted in potassium phosphate buffer (pH 7.8) containing 0.5% (w/v) PVP, 0.1 mM Na2EDTA and 10 mM 2-mercaptoethanol. MDHAR, DHAR and GR activities were assayed as in Jiménez et al. [13]. 4.5. Measurement of antioxidant metabolites 4.5.1. Glutathione The procedure used for glutathione determination was essentially that described by Floreani et al. [8]. Extracts were prepared by grinding 0.5 g fresh weight of nodules in

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1.0 ml of ice-cold 3% (w/v) sulfosalicylic acid with a mortar and pestle. Macerate was centrifuged at 15 000 × g and 4 °C. The supernatant was incubated on ice for 15 min with 0.1 volumes of 30% (w/v) 5-sulfosalicylic and centrifuged at 12 000 × g for 3 min to precipitate proteins. Oxidized glutathione content was measured after derivatization of 300 µl of the above supernatant with 5 µl of undiluted 2-vinylpyridine and 14 µl of triethanolamine at room temperature for 30 min. 4.5.2. Ascorbate Extraction for ascorbic acid was made in cold-ice 5% (w/v) metaphosphoric acid, by grinding 0.2 g of nodules in 1 ml. The homogenate was centrifuged at 12 000 × g for 15 min at 4 °C and aliquots of the supernatant were processed and injected in the high performance liquid chromatography (HPLC)–UV system for ascorbate content analysis according to Castillo and Greppin [2]. 4.6. Catalytic iron Catalytic Fe in nodule cytosol was estimated by the bleomycin assay as in Evans et al. [6]. This Fe-specific assay is based on DNA degradation by free radicals generated in the presence of ascorbic acid as a suitable reductant plus bleomycin. 4.7. Measurement of malondialdehyde Lipid peroxides of nodules were quantified by the thiobarbituric acid (TBA) test combined with HPLC to separate the (TBA)2–MDA (malondialdehyde) adduct from other components absorbing at 532 nm present in the nodules as described by Evans et al. [6]. 4.8. Measurement of oxidative damage to proteins Carbonyl groups content of protein was determined as a hallmark for oxidative damage. These were estimated by their reaction with 2, 4-dinitrophenylhydrazine to form coloured dinitrophenylhydrazones as described by Evans et al. [6].

Acknowledgements The authors are grateful to Dr. P.J. Evans and Dr. B. Halliwell for their collaboration in the catalytic iron and oxidative damage assays. The help of Dr. J.A. Hernández and Dr. F. Sevilla in measuring the activity of the enzymes of ASC–GSH cycle is greatly appreciated. We also wish to thank Dr. N.J. Briat for kindly providing the anti-ferritin

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serum, as well as C. Mesa, M.L. Melendo, M.I. Menéndez, and F. Pinto (Servicio de Microscopía, C.C.M.A., Madrid) for their technical assistance. M.J.H.J. and M.M.L. were recipients of a fellowship and a contract from Ministerio de Educación y Cultura (Spain), respectively.

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