Reduced mitochondrial and ascorbate–glutathione activity after artificial ageing in soybean seed

Journal of Plant Physiology 171 (2014) 140–147

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Physiology

Reduced mitochondrial and ascorbate–glutathione activity after artificial ageing in soybean seed Xia Xin a , Qian Tian a,b , Guangkun Yin a , Xiaoling Chen a , Jinmei Zhang a , Sophia Ng c,d , Xinxiong Lu a,∗ a

National Genebank, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Shandong Center of Crop Germplasm Resources, Jinan 250100, China c Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, 310058 Hangzhou, China d Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, WA, Australia b

a r t i c l e

i n f o

Article history: Received 4 March 2013 Received in revised form 2 September 2013 Accepted 2 September 2013 Available online 21 November 2013 Keywords: Artificial ageing Ascorbate–glutathione cycle Mitochondria Soybean seed

a b s t r a c t The effect of artificial ageing on the relationship between mitochondrial activities and the antioxidant system was studied in soybean seeds (Glycine max L. cv. Zhongdou No. 27). Ageing seeds for 18 d and 41 d at 40 ◦ C reduced germination from 99% to 52% and 0%, respectively. In comparison to the control, malondialdehyde content and leachate conductivity in aged seeds increased and were associated with membrane damage. Transmission electron microscopy and Percoll density gradient centrifugation showed that aged seeds mainly contained poorly developed mitochondria in which respiration and marker enzymes activities were significantly reduced. Heavy mitochondria isolated from the interface of the 21% and 40% Percoll were analyzed. Mitochondrial antioxidant enzymes activities including superoxide dismutase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were significantly reduced in aged seeds. A decrease in total ascorbic acid (ASC) and glutathione (GSH) content as well as the reduced/oxidized ratio of ASC and GSH in mitochondria with prolonged ageing showed that artificial ageing reduced ASC–GSH cycle activity. These results suggested an elevated reactive oxygen species (ROS) level in the aged seeds, which was confirmed by measurements of superoxide radical and hydrogen peroxide levels. We conclude that mitochondrial dysfunction in artificially aged seeds is due to retarded mitochondrial and ASC-GSH cycle activity and elevated ROS accumulation. © 2013 Elsevier GmbH. All rights reserved.

Introduction Seed ageing leads to seed deterioration. In agricultural production, aged seeds lead to commercial and genetic losses. Two common storage methods to limit seed deterioration are to reduce the moisture content and storage temperature (Walters et al., 2004; Lu et al., 2005). Even under optimal storage conditions, however, seeds suffer a variety of biochemical and metabolic alterations,

Abbreviations: APX, ascorbate peroxidase; ASC, ascorbic acid; ASH, Reduced ascorbate; ATP, adenosine triphosphate; BSA, bovine serum albumin; COX, cytochrome c oxidase; DHA, dehydroascorbate (oxidized ascorbate); DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; MDA, malondialdehyde; MDHAR, monodehydroascorbate reductase; RCR, respiration control rate; ROS, reactive oxygen species; SOD, superoxide dismutase; TCA, tricarboxylic acid; TEM, transmission electron microscopy; TES, N-Tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid. ∗ Corresponding author at: Zhongguancun South Street, Haidian District, Beijing 100081, China. Tel.: +86 10 62174099; fax: +86 10 62114309. E-mail address: [email protected] (X. Lu). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.09.016

including lipid peroxidation, enzyme inactivation, and disruption of cellular membranes (Rajjou et al., 2008; Sveinsdóttir et al., 2009; Kaewnaree et al., 2011; Hu et al., 2012). Understanding the mechanisms of seed ageing will lead to new methods for seed conservation and longevity. Superoxide radical (O2• ¯) and hydrogen peroxide (H2 O2 ) are reduce seed vigour (Bailly et al., 2008) and the accumulation of reactive oxygen species (ROS) is the main contributor to seed deterioration (Bailly et al., 2008; Rajjou et al., 2008; Oracz et al., 2009; Bailly and Kranner, 2011; Bellani et al., 2012; Hu et al., 2012; Yao et al., 2012). The antioxidative system prevents ROS accumulation in seed ageing (Bailly et al., 2002; Pukacka and Ratajczak, 2005, 2006; Yao et al., 2012) and several studies have revealed a close relationship between seed deterioration and a reduction in the activity of various antioxidative systems in soybean (Sung, 1996), cotton (Goel et al., 2003), sunflower (Kibinza et al., 2006), pea (Yao et al., 2012), and elm (Hu et al., 2012) seeds. The mitochondrion is the major site for production and scavenging of ROS during seed germination (Bewley, 1997; Møller, 2001; Howell et al., 2006; Navrot et al., 2007) and mitochondrial

X. Xin et al. / Journal of Plant Physiology 171 (2014) 140–147

biogenesis and restoration provide energy compounds and intermediates for cellular biosynthesis (Logan et al., 2001; Howell et al., 2006; Macherel et al., 2007; Taylor et al., 2010; Carrie et al., 2013). Mitochondria are also important in stress tolerance (Stupnikova et al., 2006; Roschzttardtz et al., 2009; Smiri and Chaoui, 2009). However, the role of the mitochondria in seed ageing and ROS accumulation has not been thoroughly described. In this work, we analyzed the physiological deterioration of artificially aged seed by studying mitochondrial ultra-structure, enzymatic activity, respiration, the antioxidative enzymatic defense system, and cellular ROS accumulation. Materials and methods

141

and 1 mM DTT). The homogenate was centrifuged for 5 min at 2000 × g and the resulting supernatant was centrifuged for 10 min at 20,000 × g. The pellet was resuspended in wash buffer (0.3 M sucrose, 1 mM EGTA, and 10 mM MOPS/KOH, pH 7.2) and spun at low speed (2000 × g) for 15 min followed by high-speed centrifugation (20,000 × g) for 15 min. The pellet was re-suspended in a small volume of wash buffer [0.3 M mannitol, 0.5% (w/v) BSA and 10 mM N-Tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES; pH 7.5)] and loaded on a Percoll step gradient (from bottom to top: 1 volume 40% and 1 volume 21% Percoll in wash buffer). The gradient was centrifuged at 40,000 × g for 1 h. The mitochondrial fraction located at the interface between the 40% and 21% layers was aspirated and washed 3 times in wash buffer before respiratory activity measurements.

Plant material and treatment Respiratory activity measurements in isolated mitochondria Soybean (Glycine max L. cv. Zhongdou No. 27) seeds were obtained from the National Genebank of China. The seed germination percentage was 99% and moisture content was 12.6%; storage was at −20 ◦ C. Seeds were artificially aged by sealing them in an air-tight aluminium foil bag for 0, 18 d, 41 d at 40 ◦ C. After treatment, the seeds were stored at −20 ◦ C. Prior to each experiment, the frozen seeds were transferred to 25 ◦ C for 2 d in an aluminium foil bag. For germination analysis, seeds were incubated for 7 d in an artificial climate incubator at 25 ◦ C in the dark as described by the International Seed Testing Association (ISTA, 1996). Germination percentage was measured on the 7th day after imbibition. To measure seedling dry weight, seedlings without cotyledons were dried at 103 ◦ C for 5 h. Electric conductivity Ten whole dry seeds were soaked in 25 mL Milli-Q water (Millipore, Milford, MA, USA) at 25 ◦ C. The electric conductivity of the seed leachate was determined at regular intervals with an electrical conductivity metre (Delta 326, METTLER-TOLEDO, Switzerland). Absolute conductivity was measured after treating the seeds with boiling water for 30 min. Results were presented as relative electric conductivity, i.e. electric conductivity at different soaking times relative to the absolute conductivity. Lipid peroxidation Axes collected from 24 h-imbibed soybean seeds were ground at 4 ◦ C. Lipid peroxidation was determined as the concentration of malondialdehyde (MDA) according to Heath and Packer (1968) and Hendry et al. (1992). This assay was based on thiobarbituric acid. Transmission electron microscopy Axes collected from 24 h-imbibed soybean seeds were cut into slices and fixed immediately with 2.5% (v/v) glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2). Each sample was dehydrated with a graded ethanol series and embedded in Spurr resin. Ultra-thin sections were prepared with a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with uranyl acetate followed by lead citrate prior to observation. The sections were observed and photographed with a Jeol JEM-1230 transmission electron microscope (TEM, Kyoto, Japan). Mitochondrial isolation Axes collected from 24 h-imbibed soybean seeds were ground at 4 ◦ C in extraction buffer (0.3 M sucrose, 5 mM tetrasodium pyrophosphate, 10 mM KH2 PO4 , pH 7.5, 2 mM EDTA, 1% [w/v] PVP40, 1% [w/v] bovine serum albumin (BSA), 5 mM cysteine,

All oxygen consumption measurements were performed using a Clark-type oxygen electrode (Chlorolab2, Hansatech Instrument, UK) at 25 ◦ C. Mitochondria equivalent to 100 ␮g mitochondrial protein were suspended in 1 mL respiration buffer (0.3 M sucrose, 5 mM KH2 PO4 , 10 mM TES, 10 mM NaCl, 2 mM MgSO4 , and 0.1% [w/v] BSA, pH 6.8). Succinate (10 mM), NADH (1 mM), and ADP (0.8 mM) were added as required (Millar et al., 2001). Cytochrome c oxidase (COX) (EC 1.9.3.1) activity in isolated mitochondria was measured as a decrease in absorbance at 550 nm (ε550 = 13.5 mM−1 cm−1 ) and 25 ◦ C due to cytochrome c oxidation as described by Neuburger, 1985. Mitochondrial malate dehydrogenase (MDH) (EC 1.1.1.37) activity was determined by monitoring the increase in absorbance at 340 nm (ε340 = 6.2 mM−1 cm−1 ) and 25 ◦ C due to NADH production as described by Glatthaar et al. (1974). Antioxidative enzyme activities in isolated mitochondria All antioxidative enzymes were extracted from purified mitochondria isolated from soybean embryonic axes after 24 h imbibition. All extraction procedures were carried out at 0 to 4 ◦ C. Measurements were performed as follows: Superoxide dismutase (SOD) (EC 1.15.1.1) activity was assayed by monitoring inhibition of the photochemical reduction of nitro blue tetrazolium at 560 nm and 25 ◦ C as described by Beyer and Fridovich (1987). Ascorbate peroxidase (APX) (EC 1.11.1.7) activity was measured as the decrease in absorbance of ASC at 290 nm (␧290 = 2.8 mM−1 cm−1 ) and 25 ◦ C due to oxidation by H2 O2 as described by Nakano and Asada (1981). Glutathione reductase (GR) (EC 1.6.4.2) activity was determined by the decrease in absorbance at 340 nm (ε340 = 6.2 mM−1 cm−1 ) and 25 ◦ C due to NADPH oxidation, as described by Madamanchi and Alscher (1991). Dehydroascorbate reductase (DHAR) (EC 1.8.5.1) activity was measured according to Dalton et al. (1993) by monitoring the increase in absorbance at 265 nm (␧265 = 14 mM−1 cm−1 ) and 25 ◦ C caused by DHA formation. Monodehydroascorbate reductase (MDHAR) (EC 1.6.5.4) activity was assayed as described by Arrigoni et al. (1992) by monitoring the decrease in absorbance at 340 nm (␧340 = 6.2 mM−1 cm−1 ) and 25 ◦ C due to NADH oxidation. Determination of ASC and glutathione (GSH) in isolated mitochondria ASC and GSH in purified mitochondria were extracted with icecold 5% sulfosalicylic acid. After vortex-mixing for about 5 min, it was centrifuged at 20,000 × g for 20 min at 4 ◦ C, and the content of ASC and GSH in the supernatant was measured. Reduced ascorbate (ASH) and dehydroascorbic acid (DHA) were determined as described by Law et al. (1983). Reduced glutathione (GSH) and

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100

A

Relative electrolyte leakage (%)

Germination percentage (%)

100

80

60

40

20

0 1

2

3

4

5

6

A 80

60

40

20

0

7

0

Germinating time (days)

5

10

15

20

25

Time (h)

50

16

B 14

40

MDA Content (umol g-1 FW)

Seedling weight (mg DM)

0d 18 d 41 d

30

20

10

a

B

12 b 10

b

8 6 4 2

0 0d

18 d

41 d

0 0d

Aging treatment

18 d

41 d

Aging treatment Fig. 1. Changes in seed vigour after artificial ageing. (A) Germination of control (0 d, 䊉) seeds and seeds aged for 18 d () and 41 d (). (B) The dry weight of seedlings developed from seeds aged for 0 d, 18 d, and 41 d. Data represent the mean ± standard error of 4 independent experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 4).

oxidized glutathione (GSSG) content were determined according to Griffith (1980) by the 5,5 -dithiobis-(2-nitrobenzoic acid)-GR recycling procedure. Determination and detection of ROS Hydrogen peroxide (H2 O2 ) content in seed axes collected from 24 h imbibed soybean seeds was determined by absorbance at 410 nm as described by Matsubara et al. (1983). The rate of superoxide radical (O2 • ¯ ) generation was determined according to Elstner and Heupel (1976). Seed axes were isolated and transversely cut into thin slices. The tissues were stained and imaged with a fluorogenic dye kit (Molecular Probes, Invitrogen, USA) as described by Miller et al. (2009). Protein concentration assay Protein concentrations were determined as described by Bradford (1976), using BSA standard. Statistical analyses Correlation analysis was performed with SPSS Correlate (Bivariate, two-tailed). Mean separations were performed with one-way

Fig. 2. Changes in the seed cell membrane after artificial ageing. (A) Electric conductivity of seed leachate during a 24-h imbibition time course of seeds aged for 0 d (䊉), 18 d (), and 41 d (). (B) Accumulation of malondialdehyde (MDA) as the product of lipid peroxidation in embryonic axes of 24 h-imbibed seeds after ageing for 0, 18 d, and 41 d. Data represent the mean ± standard error of 4 independent experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 4).

ANOVA and differences at p < 0.05 were considered significant. Results were pooled across repeated experiments.

Results Artificial ageing leads to vigour loss Seeds were stored in a sealed aluminium foil bag for 0 d, 18 d, and 41 d at 40 ◦ C. The germination percentage of seeds artificially aged for 18 d and 41 d was 52% and 0%, respectively, in comparison to untreated seeds (Fig. 1A). The dry weight of the aged seedlings was also significantly reduced (Fig. 1B), indicating that artificial ageing at 40 ◦ C dramatically reduced seed vigour, affecting the rate and homogeneity of seed germination. Leachate conductivity and the content of MDA equivalents were negatively correlated to seed viability, as the Pearson correlation between viability and conductivity was −1.0, and that between viability and MDA was −0.976. Conductivity was higher in artificially aged seed than in the control (Fig. 2A) and increased with longer ageing duration. Compared to the control, MDA content increased by 20% and 72% in seeds artificially aged for 18 d and 41 d,

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Fig. 3. Transmission electron micrographs of the ultra-structure of axes from seeds aged for 0 d (A and B), 18 d (C and D), and 41 d (E and F). Overview of cells aged for 0, 18 d, and 41 d (A, C and E). Ultra-structure of mitochondria from seeds aged for 0, 18 d, and 41 d (B, D and F). M, mitochondria; Ma, matrix; Cr, cristae; V, vacuole; N, nucleus; CW, cell wall.

respectively (Fig. 2B). These results suggested that artificial ageing affected cell membrane integrity and caused oxidative damage. To understand the effects of artificial ageing on cellular ultrastructure, large organelles and other cytoplasmic inclusions were analyzed by TEM (Fig. 3). A normal cell in the control sample after 24 h imbibition contained nucleus, mitochondria, and cell wall (Fig. 3A and B). In contrast, 18 d and 41 d aged soybean embryonic axes showed serious alterations (Fig. 3 C and E). Mitochondria were typically observed in the control, with numerous well-developed cristae with easily distinguishable outer and inner boundary membranes (Fig. 3B). In contrast, mitochondria from aged samples were slightly diminished in or lacking the internal membrane structures (Fig. 3D and F). Thus, mitochondrial structure was seriously affected by artificial ageing, which caused serious defects in mitochondrial function during soybean seed imbibition. Mitochondrial activity response to artificial ageing To characterize mitochondrial biogenesis and development in response to artificial ageing, mitochondria were extracted from embryonic axes by Percoll gradient after 24 h imbibition at 25 ◦ C. Mitochondria from embryonic axes showed several COX activity peaks, which formed at the upper 21% of Percoll, indicating light mitochondria, and the interface of the 21% and 40% Percoll, indicating heavy mitochondria. This corresponded to peak COX activity, a mitochondrial marker enzyme (Fig. 4A). Although in distinct proportions, these more prominent peaks were detected in all treatments. The proportion of heavy mitochondria was significantly greater in the control than in aged seed. In comparison, the proportion of light mitochondria was greatest in the 41 d

aged seeds. These results indicated that artificial ageing inhibited mitochondrial biogenesis and restoration during soybean seed imbibition. To gain a better understanding of the effects of artificial ageing on mitochondrial activity, heavy mitochondria were isolated from fractions15–18. Membrane integrity of the purified mitochondria was measured using COX activity as an indicator in the presence or absence of triton X-100. Mitochondrial membranes retained high integrity (97%) in the control sample (Fig. 4B), but decreased to 80% and 76% in response to ageing for 18 d and 41 d, respectively (Fig. 4B). Mitochondrial membrane integrity is key factor to mitochondrial function. To investigate the effects of artificial ageing on mitochondrial functions such as the tricarboxylic acid (TCA) cycle and the electron transport chain, MDH, COX, and respiration were measured in heavy mitochondria (Fig. 4 C and D, Table 1). Compared to the control, mitochondria isolated from 41 d-treated seeds showed a 16% and 20% decrease in COX and MDH activities, respectively. Measurements of respiration rate and respiration control rate (RCR) in isolated mitochondria reflected NADH and succinate-dependent O2 consumption under stress (Table 1). In comparison to the control, mitochondria isolated from 18 d-treated seeds showed a 71.9% and 57.2% decrease in NADH and succinatedependent O2 consumption, respectively, while the RCR for NADH and succinate-dependent O2 consumption decreased by 61.1% and 24.1%, respectively. Moreover, by 41 d of treatment, mitochondria exhibited a 92.7% and 90.2% decrease in the capacity of NADH and succinate-dependent O2 consumption, while the RCR for NADH and succinate-dependent O2 consumption decreased by 74.1% and 44%. These results indicated that mitochondria were dysfunctional during aged seed imbibition. Artificial ageing inhibited restoration of

X. Xin et al. / Journal of Plant Physiology 171 (2014) 140–147

A 15

0d

10

COX activity ( as % of total activity)

5

0 15

18 d

10

5

0 15

41 d

10

5

0 5

10

15

20

25

COX activity MDH activity Mitochondria integrity (%) (umol mg-1 protein min-1) (umol mg-1 protein min-1)

144

100

a

B b

b

80 60 40 20 0 1.5

C a

1.0 b

.5

c

0.0 3

D a

b

2 1

c

0

30

0d

Fraction number

18 d

41 d

Aging treatment

Fig. 4. The purification of mitochondria by 21%:40% Percoll density gradients (A), integrity of the mitochondrial membrane (B), and the activities of cytochrome c oxidase (COX, C) and malate dehydrogenase (MDH, D) in purified mitochondria from 0, 18 d and 41 d aged seeds. COX activity was used as indicator for the presence of mitochondria. Fractions 15–18 were heavy mitochondria and fractions 2–14 were light mitochondria. Data represent the mean ± standard error of 3 independent experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 3).

60

20 c 0

C

MDHAR activity

a .15 .10 b

.05 0.00 60

GR activity

APX activity

b

c

E

a

B

a .2

b

.1

b

0.0 a

DHAR activity

SOD activity

40

.3

A

a

ab

.4

D b

.2

0.0 0d

18 d

41 d

Aging treatment 40

b c

20

0 0d

18 d

41 d

Aging treatment Fig. 5. Activities of enzymatic antioxidative system, SOD (A), APX (B), MDHAR (C), DHAR (D), and GR (E) in purified mitochondria from 0, 18 d and 41 d aged seeds. SOD, U mg−1 protein; APX, ␮ mol; ASA, mg−1 protein min−1 ; MDHAR, ␮ mol NADH mg−1 protein min−1 ; DHAR, ␮ mol ASA mg−1 protein min−1 ; GR, ␮ mol NADPH mg−1 protein min−1 . For SOD activity, one unit is defined as the amount required for 50% inhibition of the photoreduction of nitro blue tetrazolium. Data represent the mean ± standard error of 3 independent experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 3).

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Table 1 Rate of oxygen uptake (nmol O2 min−1 mg−1 protein) by mitochondria with different substrates. Substrate

0d −ADP

NADH SUCC

18 d +ADP

117.9 ± 13.7 225.7 ± 17.2 83.1 ± 11.9 136.4 ± 17.8

41 d

RCR

−ADP

+ADP

RCR

−ADP

+ADP

RCR

5.4 ± 0.2 2.5 ± 0.1

35.2 ± 5.2 36.1 ± 5.8

63.2 ± 5.3 58.4 ± 8.1

2.1 ± 0.1 1.9 ± 0.2

11.6 ± 2.8 15.6 ± 1.3

16.4 ± 2.6 13.3 ± 2.3

1.4 ± 0.1 1.4 ± 0.1

Note: The rate of O2 consumption was measured in the presence of NADH or succinate (SUCC) without (−ADP) or with ADP (+ADP). The respiration control rate (RCR) was the ratio of respiration rate with ADP and after exhaust of ADP. Values represented the means of three separate experiments ± SD.

To investigate the effect of artificial ageing on ROS production, ROS accumulation was determined by spectrophotometry and a fluorogenic dye kit (Fig. 7). By 18 d of treatment, the accumulation of O2 • ¯ and H2 O2 was higher than in the control (Fig. 7A). The results of the fluorogenic dye kit were consistent (Fig. 7B). As shown in Fig. 7B, the fluorescent signal was increased in 18 d-aged seeds. By 41 d of treatment, cellular ROS was similar to the control. We presume that mitochondrial activities were deeply reduced in 41 d-aged seed, which might lead to the decrease in ROS production. Discussion In practice, storing seeds at low temperature, with low moisture content, is the ideal way to inhibit seed deterioration. However, many reports have indicated that seeds age slowly and vigour is reduced or lost by long-term storage (Walters et al., 2004; Lu et al., 2005). To better understand the mechanism of seed ageing, artificial ageing is often used to qualify seed vigour (Goel et al., 2003; Rajjou et al., 2008). In this study, soybean seed vigour was altered by storage at 40 ◦ C for up to 41 d. Seed vigour rapidly declined from 99% to 0% (Fig. 1). Furthermore, the reduction in seed vigour caused a significant increase in the conductivity of leachate and MDA contents (Fig. 2), suggesting that artificial ageing affects the integrity of cell membranes during soybean seed imbibition at 25 ◦ C. Rapid resumption of the membrane system in dry seeds, especially for

A

ASH DHA ASA/DHA

6

16

12 4 8

2

ASH/DHA

Accumulation of cellular ROS in aged seeds

8

4

0

0 0d

18 d

41 d

Aging treatment 16

1.6

B

GSH GSSG GSH/GSSG

1.2

12

.8

8

.4

4

0.0

GSH/GSSG

To assess the effect of artificial ageing on the mitochondrial antioxidative system, we determined the activities of ROSscavenging enzymes (SOD, APX, MDHAR, DHAR, and GR) and the contents of non-enzymatic antioxidants (ASC and GSH) in mitochondria. Artificial ageing significantly affected the activities of ROS scavenging enzymes (Fig. 5). In comparison to the control, mitochondrial SOD, APX, MDHAR, DHAR, and GR activities decreased by 80%, 77%, 86%, 8%, and 67% in 41 d-treated seeds (Fig. 5). Thus, the capacity of the enzymatic antioxidative system in isolated mitochondria was reduced by artificial ageing. To further investigate the effect of artificial ageing on the mitochondrial ASC–GSH cycle, we analyzed the contents of reduced ascorbate (ASH), oxidized ascorbate (DHA), reduced glutathione (GSH), and GSSG in isolated mitochondria. The reduced/oxidized forms of both substances decreased in mitochondria with prolonged ageing treatment (Fig. 6). Although DHA increased with prolonged ageing, total ASC decreased (Fig. 6A). These changes were consistent with a prominent reduction in ASH/DHA ratio (Fig. 6A). Moreover, the GSH/GSSG ratio decreased quickly, as did the total GSH pool (Fig. 6B). The reduction of both ratios indicated that aged seeds were less able to scavenge ROS.

ASH and DHA content (nmol mg-1 protein)

Enzymatic antioxidative system in mitochondria after artificial ageing

mitochondria, is critical for seed vigour (Bewley, 1997; Taylor et al., 2010; Carrie et al., 2013). It is usual for dry seeds to contain poorly differentiated mitochondria, and after imbibition mitochondria will be filled with sufficient TCA cycle enzymes and terminal oxidases to provide adequate amounts of adenosine triphosphate (ATP) and intermediates for cellular biosynthesis (Logan et al., 2001; Howell et al., 2006; Macherel et al., 2007; Taylor et al., 2010; Carrie et al., 2013). In comparison to the control, the mitochondria of 18 d and 41 d aged seeds were severely altered in their morphology (Fig. 3B, D and F). The membrane structure was destroyed as the number of cristae gradually fell to zero, and the matrix became less dense (Fig. 3D and F). Furthermore, our studies confirmed that mitochondrial biogenesis during seed imbibition was inhibited by artificial ageing. Mitochondrial ultra-structural damage, which occurred in response to artificial ageing, negatively impacted mitochondrial functions. COX and MDH are important mitochondrial marker enzymes that are positively correlated with respiration (Day et al., 2004). The gradual decrease of COX and MDH activities might result in decreased respiration associated with ageing (Fig. 4C and D). Mitochondrial membrane integrity decreased in aged seeds

GSH and GSSG content (nmol mg-1 protein)

the electron transport chain and the oxidative phosphorylation in soybean mitochondria after 24 h imbibition at 25 ◦ C.

0 0d

18 d

41 d

Aging treatment Fig. 6. The contents of ASC (A) and GSH (B), and the ratios of ASH/DHA (A) and GSH/GSSG (B) of purified mitochondria from seeds aged for 0, 18 d and 41 d. Data represent the mean ± standard error of 3 different experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 3).

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Fig. 7. Accumulation of ROS in 0, 18 d, and 41-d aged seed axes. (A) The rate of O2 • − generation (䊉) and accumulation of H2 O2 (). The data represent the mean ± standard error of 3 independent experiments. All treatments significantly differed from the control (p < 0.05, one-way ANOVA, n = 3). (B) Accumulation of ROS as demonstrated by the fluorogenic dye kit.

(Fig. 4B), which might indicate the mitochondrial membrane system was difficult to recover in aged seed during imbibition. The mitochondrial membrane plays an important role in oxidative phosphorylation (Frey and Mannella, 2000; Logan et al., 2001). In fact, since the seed axis does not contain chloroplasts during seed imbibition, ATP production in the mitochondria is the main source of energy. Artificial ageing severely affects the rate of NADH and succinate-dependent O2 consumption and RCR (Table 1). These results suggest that aged seeds possess a low capacity for the electron transport chain in comparison to the control. Ageing directly reduces the efficiency of electron transport chains, thereby reducing ATP production. This observation implied that aged seeds could not provide sufficient ATP for germination, consistent with previous reports in soybean (Benamar et al., 2003; Yin et al., 2009). The plant mitochondrial matrix contains ROS scavenging systems, including enzymatic and non-enzymatic antioxidative systems (Møller, 2001; Rhoads et al., 2006). Plant mitochondrial activities affect ROS scavenging and accumulation (Smirnoff, 1998; Macherel et al., 2007; Navrot et al., 2007). Overall, our results showed that SOD activity decreased with prolonged ageing, and similar results were observed in APX, MDHAR, and GR activities (Fig. 5). The mitochondrial ASC–GSH system possessed weaker reduced/oxidized forms in aged seeds (Fig. 6), which might lead to ROS accumulation. Thus, the aged seeds might be easily attacked by O2 •− and/or H2 O2 . Our results confirmed that ROS accumulated in aged seeds (Fig. 7). However, it is difficult to determine how much ROS in the total cellular population originated from the mitochondria (Veljovic-Jovanovic et al., 2002; Halliwell and Whiteman, 2004). This was consistent with previous studies in which ROS accumulation was positively associated with a

reduction of plant tolerance to many environmental stresses, manifested by lower survival and higher ion leakage and MDA levels. In summary, our data demonstrated a reduction in mitochondrial activities and the ASC–GSH cycle in aged seed, which might cause ROS accumulation. However, mitochondrial functions may be affected by ROS, which might cause insufficient ATP and intermediates necessary for seed germination. Acknowledgements This work was supported by the National Natural Science Foundation of China Programme (31201280) and National Key Technology R&D Programme (2013BAD01B01). References Arrigoni O, De Gara L, Tommasi F, Liso R. Changes in ascorbate system during seed development of Vicia faba. Plant Physiol 1992;99:235–8. Bailly C, Bogatek-Leszczynska R, Come D, Corbineau F. Changes in activities of antioxidant enzymes and lipoxygenase during growth of sunflower seedlings from seeds of different vigour. Seed Sci Res 2002;12:47–55. Bailly C, El-Maarouf-Bouteau H, Corbineau F. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. C R Biol 2008;331:806–14. Bailly C, Kranner I. Analyses of reactive oxygen species and antioxidants in relation to seed longevity and germination. Methods Mol Biol 2011;773:343–67. Bellani LM, Salvini L, Dell’Aquila A, Scialabba A. Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination. Acta Physiol Plant 2012;34:1789–99. Benamar A, Tallon C, Macherel D. Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing. Seed Sci Res 2003;13:35–45. Bewley JD. Seed germination and dormancy. Plant Cell 1997;9:1055–66.

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