This work was supported by the ‘948’ Project of the State Forestry Administration of China (2011-4-54) and The Special Fund Project for the Scientific Research of the Forest Public Welfare Industry (201104024).
Seed deterioration is poorly understood and remains an active area for research. Seeds of elm (Ulmus pumila L.) were aged at 37 °C above water [controlled deterioration treatment (CDT)] for various lengths of time to assess programmed cell death (PCD) and reactive oxygen species (ROS) product in embryonic tissues during a 5 d period. The hallmarks of PCD were identified in the elm seeds during CDT including TUNEL experiments, DNA laddering, cytochrome c (cyt c) leakage and enzymatic activities. These analyses indicated that PCD occurred systematically and progressively in deteriorated elm seeds. Cyt c release and increase in caspase-3-like/DEVDase activity occurred during CDT, which could be suppressed by ascorbic acid (AsA) and caspase-3 inhibitor Ac-DEVD-CHO, respectively. In situ localization of ROS production indicated that the distinct spatial-temporal signature of ROS during CDT coincided with the changes in PCD hallmark features. Multiple antioxidant elements were activated during the first few days of CDT, but were subsequently depleted as PCD progressed. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during elm seeds CDT and suggest an important role for PCD in seeds deterioration.
Seed deterioration is a major problem in agricultural production. Some damage to seeds during storage is inevitable and depends on the temperature and relative air humidity in the storage vessel, seed moisture content (MC), duration of storage, type of seed and initial seed quality (Priestley 1986). Although seed deterioration has been studied for many years, published reports typically focus on free radical-mediated lipid peroxidation, enzyme inactivation, protein degradation, disruption of cellular membranes and damage to genetic integrity (Priestley 1986; Smith & Berjak 1995; Walters 1998; McDonald 1999; Walters et al. 2006). The detailed mechanism of seed deterioration is still unclear.
Early studies described chromosome damage induced by accelerated ageing in barley and lettuce seeds (Murata, Tsuchiya & Roos 1984; Rao, Roberts & Ellis 1987). In a study of rye seeds, DNA was progressively cleaved as seeds dried, deteriorated and lost their ability to germinate (Cheah & Osborne 1978; Osborne, Sharon & Ben-Ishai 1981). DNases operating at different levels of water activity were the cause of this DNA cleavage (Boubriak et al. 2000). As seeds lost viability during ageing, DNA gradually degraded into internucleosomal fragments, resulting in ‘DNA laddering’ (Kranner et al. 2006, 2010). Seeds treated with the caspase inhibitor z-VAD-fmk exhibited extended longevity, confirming that caspase-like proteins contribute to loss of viability in seeds (Kranner et al. 2006). These findings are similar to certain characteristics of programmed cell death (PCD), which led us to suggest that seed degeneration occurs by PCD.
PCD is a mode of self-protection that can be triggered by various internal and external environmental factors. PCD is regulated by genes and involves the activation of a variety of molecular pathways. Cells that are ultimately unnecessary or damaged beyond repair are removed by this process (Reape & McCabe 2008). Studies have shown that the death of plant cells induced by ozone (Overmyer et al. 2005), salinity (Andronis & Roubelakis-Angelakis 2010), pathogens (Hatsugai et al. 2004), heat stress (Zuppini, Bugno & Baldan 2006) and ultraviolet (UV) radiation (Danon et al. 2004) exhibit some fundamental processes comparable to animal apoptosis, including release of cytochrome c (cyt c), activation of specific proteases and DNA fragmentation.
Whereas PCD has been well studied in animals, scientific knowledge is limited regarding the mechanisms that regulate and execute plant cell death (Cacas 2010; Pinto, Locato & Gara 2012). Hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) are recognized as key modulators of PCD and many other biological processes including growth, development and stress adaptation (Yao et al. 2002; Gechev et al. 2006; Lin, Wang & Wang 2006; Gao et al. 2008). Tobacco (Nicotiana tabacum) cells undergoing PCD exhibit an immediate superoxide anion (O2-) and H2O2 burst, but death can be prevented with antioxidants such as ascorbic acid (AsA) and superoxide dismutase (SOD; Vacca et al. 2004). The accumulation of ROS is often indicated as the prime cause of seed deterioration because ROS initiate reactions with polyunsaturated fatty acids, which leads to lipid peroxidation, destruction of cellular membranes, and damage to proteins and nucleic acids (McDonald 1999; Bailly et al. 2000; Bailly 2004; Bailly, Ei-Maarouf-Bouteau & Corrbineau 2008). However, the literature is limited regarding PCD cues during seed deterioration. Studies of lettuce, rye, rice, peanuts and maize have indicated that cellular organelles of aged seeds exhibit considerable irregularity and distortion (Priestley 1986). It is not clear, however, whether these phenomena are associated with PCD.
Controlled deterioration treatment (CDT) was developed as an alternative for vigour testing in seeds, to provide greater precision in the control of the high relative humidity (RH) and temperature to which seeds are exposed during the accelerated ageing test. During CDT, water uptake by seeds of each sample occurs at different speeds, and thus, the initial seed MC in the CDT is adjusted to the same level, before exposure to a high temperature in a water bath (McDonald 1999; Rajjou et al. 2008). In this study, elm seeds that had undergone CDT (37 °C, 100% RH) were examined to determine the involvement of PCD events, detected by cytological and molecular analyses. Our results suggest that PCD occurs systematically at specific tissue sites in the embryo and times during seed deterioration. In addition, we determined the production and location of H2O2 that occur during CDT-induced seed PCD. To our knowledge, this is the first comprehensive report of a direct link between seed deterioration and PCD, based on multiple factors occurring with the generation of H2O2.
MATERIALS AND METHODS
Seeds of elm (Ulmus pumila L.) were collected from the Beijing Forestry University Campus, China, in May 2011. The original germination rate was 98%, and the MC was 0.077 g H2O g−1 dry weight (DW). Before the experiments, the seeds were stored at −20 °C in tightly closed containers.
Controlled deterioration test
The seeds were surface sterilized using 5% sodium hypochlorite solution for 10 min, and rinsed with distilled water three times. After such treatment, seeds were rapidly air dried until they reached their original MC. The seeds that were equilibrated in sealed bottles at 37 °C above water for 1 d are defined as the post-equilibration (PE) and day 0 as control, then began the CDT until viability loss (5 d) as judged by germination tests. Samples were taken at 1 d intervals to investigate characteristics of PCD.
The seeds were treated with AsA (10 mmol) and Ac-DEVD-CHO (100 mmol) or water for 12 h without the radicle outstretched and then redried to the original MC under air and then aged until the untreated seeds had lost 50% viability (3 d).
We initially tested the effect of AsA and Ac-DEVD-CHO on seeds CDT over a range of concentrations (0, 10, 25, 50, 100 and 200 mmol), then chose the optimum concentration for inhibition.
Determination of MC
The MC of elm seeds was determined gravimetrically (103 °C for 17 h). The seeds were sampled for each determination. The MC of seeds was expressed as g H2O g−1 DW. The values shown were obtained in three replicates.
Four 12 cm Petri dishes with double-layer wet filter paper were used as germinating beds, and 50 seeds were added to each Petri dish and germinated at 25 °C. A seed was considered as germinated when the radicle had elongated to 2–3 mm. The rate of germination was recorded daily, and the radicle DW was determined after 7 d. The vigour (VI) was calculated using the following formula:
where Gt represents germination at time t and SX represents the average radicle weight after germinating.
Twenty seeds of elm in three replications were rinsed several times with ddH2O, surface dried with filter paper and soaked in 20 mL of deionized water at 25 °C for 24 h. Electrolyte leakage measurements were performed using an automatic seed conductivity meter (DDS-307, China) at regular intervals. At the end of the experiment, the maximum electrolyte leakage was measured after killing the seeds by boiling them for 30 min in sealed test tubes and, then, incubating them in a water thermostat at 25 °C for 1 h. Electrical conductivity was quantified in µS cm−1 and calculated per 1 seed. In addition, leakage was calculated as a percentage of the greatest leakage measured after seed killing by heating.
Light microscope observation
Samples were immediately fixed in formaldehyde:glacial acetic acid:70% ethyl alcohol (FAA, 5:5:90) for 24 h. The fixed material was placed in different concentrations of ethanol and incubated for 2 h (ethanol was replaced after 1 h). The seeds were placed in a 50% xylene:50% ethanol solution after being removed from 100% ethanol and then were hyalinized by passing through a graded xylene series for 2 h each. Next, trivial wax was added into the material, which was incubated at 38 °C overnight. The fixed material was placed in a small cup with liquid wax and incubated at 56 °C, and the liquid wax was replaced every 4 h. The material (with paraffin) was poured into previously prepared cardboard boxes and arranged using a hot needle. Then, the material was placed into cold water after congealing at 4 °C overnight. Wrapping material was cut into small pieces of wax, and then samples were sliced to 8 µm sections by using a microtome. Sections were glued to polylysine-coated slides with protein patches, and then some distilled water was added to the coverslip preparation, which was dried in a water bath at 45 °C. Sections were stained with 0.5% hematoxylin. Elm seeds with different degrees of deterioration were observed under an Olympus microscope, and photomicrographs were taken.
Ultrastructure determined via transmission electron microscopy
The seed hypocotyls were removed and placed into 2.5% glutaraldehyde fixative (0.16 mmol L−1 phosphate buffer preparation). Sections were cut into 2–4 mm pieces in each of the fixatives and placed on a vacuum pump for 3 h. The samples were stored in the refrigerator overnight. All of the sections were post-fixed for 1 h in a 1% solution of osmium tetroxide in 0.1 m sodium phosphate buffer, pH 7.3. They were dehydrated in graded alcohol solutions and embedded in a mixture of Epon and Araldite. Ultrathin sections were counterstained with uranyl acetate and lead citrate before observation with an electron microscope.
In situ detection of DNA fragmentation (TUNEL assay)
The seeds were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4). Samples were dehydrated in a graded ethanol series and then embedded in paraffin. Sample sections (8 µm) mounted on poly-l-lysine-coated glass slides were processed with the in situ Cell Death Detection Kit AP according to the manufacturer's instructions (Roche, Germany).
DNA extraction and analysis
Genomic DNA was isolated from approximately 50 mg freeze-dried seed powder using cethyltrimethylammonium bromide (CTAB) and quantified spectrophotometrically at 260 nm (Stewart & Via 1993). DNA quantity and quality were assessed spectrophotometrically at 260, 280 and 230 nm. About 10 µg of DNA was separated on 1.5% agarose gel, stained with ethidium bromide, visualized using a UV transilluminator (Syngene) and the intensity of the 180 bp fragments quantified by image analysis, using the Syngene image analysis programme.
DNA extraction from microbial contaminants and PCR amplification of the internal transcribed spacer region
To investigate whether fungal contamination of seeds was affecting our results, it was tested if fungus-specific DNA regions could be amplified in DNA isolated from deteriorated seeds. According to Kranner et al. (2011) with small modified, to obtain fungal biomass, we picked up little hyphae from 5 d of deteriorated seeds into the 1 mL distilled water and then took 10 µL diluents daubing into the potato dextrose agar (PDA) solid culture medium, 28 °C incubation for 2 d. DNA was extracted using CTAB and quantified spectrophotometrically at 260 nm. For both seed and fungal DNA samples, PCR amplification was conducted of the internal transcribed spacer (ITS) of the ribosomal DNA repeat cluster, which is typically sequenced for molecular classification of fungi. The universal primer pair ITS1-ITS4 was first tested (5′-TCCGTAGGTGAACCTGCGG-3′ for ITS1 and 5′-TCCTCCGCTTATTGATATGC-3′ for ITS4; White et al. 1990); it can co-amplify angiosperm DNA but did produce bands when seed DNA was used as a template (data not shown). For specific detection of fungal contamination, the primer combination ITS1F-ITS4 was chosen that amplifies fungal DNA but excludes angiosperm DNA (5′-CTTGGTCATTTAGAGGAA GTAA-3′ for ITS1F). To test if the seed DNA was a suitable template for gene expression experiments, PCR amplification of the 18S gene was conducted as a control.
Detection of cyt c in seed extracts
Mitochondria and cytosolic fractions of elm seed were isolated from the control and deteriorated seeds that were imbibed at 4 °C for 24 h. Cytoplasmic fractions were isolated from the seeds as described by Balk, Leaver & McCabe (1999). About 20 µg of cytosolic or mitochondrial proteins were loaded onto a 15% sodium dodecyl sulphate (SDS)-polyacrylamide gel, separated, and transferred to a polyvinylidene difluoride membrane that was subsequently probed with the monoclonal anti-cyt c antibody (1:500 dilution; Pharmingen). The membrane was stripped, blocked in PBS containing 0.1% (w/v) Tween 20 (PBS-T) + 5% (w/v) dried skimmed milk and reprobed with a purified goat polyclonal antibody against fumarase (1:500; OriGene, Rockville, MD, USA) in block buffer at 4 °C overnight. After washing (three times for 5 min each) in block buffer, the membrane was incubated in goat anti-rabbit horse radish peroxidase (HRP) conjugate (1:10 000; Amersham, Chiltern Hills, UK) in PBS-T for 1 h at room temperature and washed. Labelling was detected by chemiluminescence (NEN, Boston, MA, USA) according to the supplier's manual. The signal was quantified using scanning densitometry of the exposed film with the exposure time adjusted to non-saturating conditions.
Measurement of caspase-3/DEVDase activity in elm seeds
Control and deteriorated seeds were homogenized in a pestle and mortar in liquid nitrogen, then added with extraction buffer [50 mm Hepes, pH 7.4, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 3 mm DL-dithiothreitol (DTT), 0.1 mm ethylenediaminetetraacetic acid (EDTA), 2 mm phenylmethanesulfonyl fluoride (PMSF)]. The homogenate was centrifuged at 15 000 g for 30 min at 4 °C, and the extract was tested for caspase-3 activity. About 15 µg of seed protein extracts were mixed with 100 µm of the indicated fluorogenic substrate Ac-DEVD-AMC, and caspase assay buffer (50 mm Hepes, pH 7.4, 100 mm NaCl, 0.1% CHAPS, 1 mm EDTA, 10% glycerol) was added to a final volume of 200 µL. After incubation at 30 °C for 30 min, the absorbance of AMC hydrolysed from the peptide substrate was measured at 460 nm in a fluorescent microplate reader (Bio-RAD Model 550, American). The data represent the mean (± SE) of three independent measurements. For inhibitor assays, seed extracts were resuspended in assay buffer and incubated for 1 h with specific caspase-3 inhibitors (Ac-YVAD-CHO). Extracts were then incubated with the substrate DEVD-AMC (100 mm).
In situ localization of ROS
ROS production was visualized after ROS staining with H2DCFDA using confocal laser scanning microscopy (CLSM) (Oracz et al. 2009). H2DCFDA permeates cells and is hydrolysed by esterases to liberate dichlorofluorescein (DCF), which reacts with H2O2 or hydroperoxides to form a fluorescent DCF-derived compound. Thus, the H2DCFDA must not be considered as an indicator of H2O2 formation but rather as an indicator of ROS generation. Live seed will stick to the top of the base vibration slicer adding potassium phosphate buffer (pH 6.0). Seed was cut into slices in 50 µm, then the slices were taken from PBS, glued to the poly-lysine-treated glass slide, and washed several times with PBS, and then the samples were put in the dark and incubated in 20 mm potassium phosphate buffer (pH 6.0) containing 100 mm dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 min. The glass slides with slices were rinsed for 10 min in the potassium phosphate buffer solution several times. Images were acquired (excitation, 488 nm; emission, 525 nm) with CLSM using a 340/1.25 numerical aperture objective. Z-series were performed with a Z-step of 5 mm, and maximum projections of Z planes were displayed.
Determination of hydrogen peroxide content
The H2O2 content was determined according to the method described by O'Kane et al. (1996). Seeds [0.5 g fresh weight (FW)] were ground in a mortar and homogenized with 5 mL of 0.2 m perchloric acid. After 15 min of centrifugation at 13 000 g at 4 °C, the resulting supernatant was neutralized to pH 7.5 with 4 m KOH and then centrifuged at 1000 g for 3 min at the same temperature. The supernatant was immediately used for spectrophotometric determination of H2O2 at 590 nm using a peroxidase-based assay. The reaction mixture contained 12 mm 3-dimethylaminobenzoic acid in 0.375 m phosphate buffer (pH 6.5), 1.3 mm 3-methyl-2-benzothiazolinone hydrazone, 20 µL (0.25 U) horseradish peroxidase (Sigma, St. Louis, MO, USA) and 50 µL of the collected supernatant to a total volume of 1.5 mL. The reaction was started by the addition of peroxidase. Increase in absorbance at 590 nm was monitored after 5 min at 25 °C and compared with the absorbance obtained with known amounts of H2O2. The results are expressed as nmol H2O2 g−1 DW and correspond to means of the values obtained with five different extracts ± standard deviation (SD).
Antioxidant enzyme assays
All enzymes were assayed at 24–26 °C, and their activities were expressed as µmol substrate consumed or products generated per minute. SOD (EC 188.8.131.52) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium according to the method of Bailly et al. (1996). Ascorbate peroxidase (APX; EC 184.108.40.206) activity was assayed as a decrease in the absorbance at 290 nm due to AsA oxidation of following the method of Nakano & ASAda (1981). The reaction mixture contained 50 mm potassium phosphate, pH 7.0, 1 mm sodium ascorbate, 2.5 mm H2O2 and an enzyme source (c. 50 µg of protein) in a final volume of 1 mL at 25 °C. Catalase (CAT; EC 220.127.116.11) activity was determined by directly measuring the decomposition of H2O2 at 240 nm as described by Bailly et al. (1996) in 50 mm potassium phosphate (pH 7.0) containing 10 mm H2O2 and an enzyme source (c. 50 µg of protein) in a final volume of 1 mL at 25 °C. Glutathione reductase (GR; EC 18.104.22.168) activity was assayed following the disappearance of absorbance at 290 nm and the oxidation of NADPH at 340 nm (Bailly et al. 1996).
Determination of AsA and GSH
Reduced AsA and dehydroascorbic acid (DHA) content were determined according to methods of Navrot et al. (2007). Oxidized glutathione (GSSG) and reduced glutathione (GSH) content were determined according to the method described by Foyer & Noctor (2003).
Protein concentration assay
Protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.
All data were analysed using a one-variable general linear model procedure (analysis of variance) by SPSS (SPSS Inc., Chicago, IL, USA). Mean separations were performed with the least significant difference test, and differences at P < 0.05 were considered significant. Results presented were pooled across repeated experiments.
Effect of CDT on seed vigour
Seed viability (i.e. germination percentage and seed vigour) was measured in deteriorated seeds as a function of time after CDT. Seed viability did not change substantially during the first day of equilibration and/or during the first day of CDT but declined progressively as of the second day of CDT (Fig. 1A). The half-viability period (P50), that is, the time required for germination to drop to about 50%, was at 3 d and only 2% of seeds germinated after 5 d of CDT. However, the seed vigour index was almost negligible after 3 d of CDT. Deteriorated seeds exhibited some pathogenic disease during the late stages of CDT.
Although seeds were previously sterilized, the surface sterilization of seeds alone may not suffice to prevent microbial growth because fungi may already be associated with internal seed tissues when shed from the mother plant (Mycock & Berjak 1992; Whitaker, Berjak & Pammenter 2008). The seeds with a high vigour can be resistant to fungal contamination, but at the later stage of CDT, especially by the fifth day of CDT (2% germination), dead seeds with a very low vigour (2% germination) had reduced ability to resist external stress, so fungal contamination was seen in the epidermis of seeds.
The sensitivity of seeds to CDT is tightly dependent on temperature and on their MC. At a constant temperature, loss of seed viability is faster with increasing MC, a key factor in seed longevity (Priestley 1986; McDonald 1999). Dried seeds had very low MC, 0.077 g H2O g−1 DW, and water uptake was rapid during the 1 d equilibration period, reaching 0.26 g H2O g−1 DW. During the following days of CDT, MC did not change significantly (Fig. 1B).
Seed cell death is also associated with an increase in cellular membrane permeability (Priestley 1986), which can be measured in terms of ion leakage. In elm seeds, membrane leakage remained constant during the first 3 d of CDT, increasing dramatically afterwards (Fig. 1B).
Light microscopy provided an anatomical overview of deterioration in elm seeds. Viability staining of seeds revealed that embryos in control seeds were plump with deep dye and had a clear outline (Fig 1C, a, b). A less abnormal morphology (Fig 1C, c) was observed up to 1 d of CDT. However, the staining of seeds CDT for 2–5 d became less intense, the radicle and seed axis tissues could not be clearly identified (Fig. 1C, d–f). By 5 d of CDT, the radicle remained unstained with hematin, and embryos were not identifiable (Fig. 1C, g). These observations suggested gradual degradation towards cell death particularly starting at 2 d of CDT.
CDT-induced PCD in seeds
Elm seeds are dicotyledonous and lack endosperm. Light microscopy confirmed embryo differentiation into hypocotyl, radicle with a cap and shoot apex. Transmission electron micrograph (TEM) images indicated that the hypocotyl consisted of an outermost protoderm (PD), ground meristem (GM) and the innermost procambium (PC). PD cells are flat and small with large nuclei and abundant cytoplasm. PC cells are rectangular with large nuclei and exhibit remarkable vitality and potential for cell division. Nuclear membranes of living hypocotyl cells are regular with uniformly distributed chromatin (Supporting Information Fig. S1).
To confirm that CDT induces PCD, TUNEL was performed on longitudinal sections of seeds at different stages of deterioration (Fig 2). TUNEL-positive nuclei first appeared in the root cap and outer epidermis of cotyledons during deterioration. Close analysis of TUNEL-positive nuclei revealed time-specific differences in DNA fragmentation during CDT. Very few TUNEL-positive nuclei were observed in the outer epidermis of cotyledons and root caps of elm seeds after equilibration (Fig. 2A, a–d). Slightly more TUNEL-positive nuclei were observed in the outer epidermis of cotyledons after 1 d of CDT, but these nuclei were sporadic and accounted for less than 10% of the total cotyledons. In contrast, TUNEL-positive nuclei in root caps were clear and accounted for more than 20% of radicle cells. The remaining cells at this stage could still be stained with methyl green (Fig. 2A, e–h). After 2 d of CDT, approximately 90% of the epidermis cells of cotyledons exhibited signs of PCD. A few brown TUNEL-positive nuclei were detected in cotyledon mesophyll cells, with fewer TUNEL-positive nuclei on the inside than on the outside of the cotyledon. At this stage, more than 80% of the root cap cells were TUNEL positive, and a few TUNEL-positive nuclei were detected in the shoot apex. In this region, the colour was lighter yellow, resulting from the brown reaction colour being altered by methyl green counterstaining, and appeared considerably different from brown TUNEL-positive nuclei in the outside epidermis of cotyledons. We speculated that small amounts of DNA fragmentation in the cells of shoot apices occurred because we observed that green nucleoli only had surrounding light brown staining. In addition, slightly TUNEL-positive nuclei began to appear in the PD and PC cells of hypocotyls. However, PC cells were only stained with methyl green (Fig. 2A, i–l).
After 3 d of CDT (54% germination), most cells of cotyledons exhibited TUNEL-positive nuclei, and the colour of TUNEL-positive nuclei was a deeper brown. Abundant brown TUNEL-positive nuclei cells were observed in the PD and GM cells of hypocotyls, and more than 80% of root cap cells appeared brown. Notably, PC cells of hypocotyls retained high cell vitality with virtually no apoptotic cells. Root-tip cells were well protected by root cap cells, and only sporadic brown apoptotic nuclei were observed (Fig. 2B, a–d). After 4–5 d (18 and 2% germination) of CDT (Fig. 2B, e–l), the cellular ultrastructure was highly disrupted, and organizational structure could not be clearly differentiated. At this stage, more than 90% of PC cells were positively labelled by TUNEL, and the cells assumed the morphology of elongated strips. However, about 10% of PC cells remained stained with methyl green at this time.
Appropriate control treatments were conducted for every set of slides (Fig. 3). We found no TUNEL-positive nuclei in the same tissues when the terminal deoxynucleotidyl transferase was omitted from the reaction (negative control; Fig. 3, a–d). In contrast, when DNaseI treatment preceded the TUNEL procedure, all nuclei in all regions of seeds (Fig. 3, e–h) were labelled by TUNEL (positive control).
A typical DNA laddering that characterizes apoptotic cell death in animals and PCD cell in some plant systems is reported here. As elm seeds lost viability, they showed the typical DNA laddering of multiples of ∼200 bp (Fig. 4). After 3 d of CDT (54% germination), the seeds began to show the same traces of DNA fragmentation, and with longer CDT, the phenomenon was more evident. To investigate if degradation of DNA during deterioration may have been caused by fungal DNases, fungal contamination was assessed using PCR with the universal fungal ITS4-ITS1F primers. DNA extracted from fungal contaminants that grew on PDA solid culture medium produced a clear band but no fungal contaminants were detected in any of the seed DNA samples. Some weak amplification may occur with highly concentrated pure DNA for some angiosperm species when ITS1F is used in combination with ITS4 (Gardes & Bruns 1993), but we did not observe any bands when seed DNA was used as a template. In contrast, the plant-specific 18S gene was not amplified when the fungal DNA was used as a template. Seed DNA produced a clear band for the 18S gene when isolated from non-aged control seeds and deteriorated seeds. DNA from dead seeds produced only a faint 18S band, indicating that after 5 d of CDT, the quality of the extracted seed DNA had deteriorated (Supporting Information Fig. S2, a,b).
Release of cyt c and caspase-3/DEVDase activity
Release of cyt c from mitochondria to cytosol is an early event in PCD (Gao et al. 2008). To investigate whether CDT-induced PCD in elm seeds involves the release of cyt c, deteriorated and control elm seeds were homogenized, and mitochondria were separated from the cytosol by centrifugation. Both cytosolic and mitochondrial fractions were examined. Proteins were isolated from each fraction and analysed by SDS–polyacrylamide gel and Western blotting using a monoclonal antibody against rat cyt c. The Western blot revealed that the mitochondria began to release cyt c to the cytosol during the second day of CDT, and cyt c could no longer be detected in mitochondria after 5 d of CDT (Fig. 5A). Western blot analysis of the cytosolic samples demonstrated that cyt c was detectable in the cytosol immediately after deterioration began and increased on the day following the treatment. To test the possibility that CDT disrupts mitochondria, we probed the membrane with an antibody against fumarase, a 50 kDa mitochondrial matrix protein. This protein remained associated with mitochondria and was only detectable at low levels in the cytosol during the late stages of CDT. These data provide additional evidence that PCD is triggered in elm seeds during CDT. Controls consistently showed no sign of cyt c in the cytosol.
To determine if cyt c was released as a result of general disruption of the outer mitochondrial membrane, we assayed outer membrane integrity using an assay based on the latency of cyt c oxidase activity. The outer membrane exhibited mitochondrial intactness values of 95–98% after the first 3 d of CDT, then decreasing to 85% during the late stages of CDT (data not shown).
To ascertain whether such a cyt c release could depend on the production of ROS caused by CDT, antioxidant AsA was added exogenously to seeds before CDT. No significant release of cyt c from mitochondria to cytosol was found in the presence of AsA during seed deterioration (Fig. 5A).
The finding that CDT-induced cyt c released from the mitochondria to the cytosol in elm seeds suggested that a caspase-like activating pathway may exist in seeds. To test this hypothesis, we used a synthetic fluorogenic substrate for animal caspase-3 (Ac-DEVD-AMC) to examine the caspase-3/DEVDase activities in extracts from aged and control seeds. We found that CDT immediately triggered a significant increase in caspase-3/DEVDase proteolytic activity. After 3 d of CDT, caspase-3/DEVDase proteolytic activity increased from 38.23 ± 7.5 to 118 ± 3.3 pmol min−1, which was about 3.1-fold greater than that of the control. From 3 d of CDT, it began to decease slowly, and decreased to 113.20 ± 4.4 pmol min−1 by 5 d of CDT, but still higher than that of control (Fig. 5B). The seeds pretreated with Ac-DEVD-CHO (a caspase-3-like inhibitor) and AsA clearly decreased the activity of caspase-3/DEVDase by 34.8 and 30.4%, respectively, compared with that of 3 d of CDT (Fig. 5C).
In the absence of Ac-DEVD-CHO and AsA, seed germination capacity at 3 d of CDT was approximately 50%. About 78 and 69% germination was observed in seeds pretreated with Ac-DEVD-CHO and AsA at the same time of CDT, respectively (Fig. 5D).
ROS production in elm seeds undergoing seed PCD
Based on the effect of AsA on germination, release of cyt c and caspase activity, we proposed that ROS produced during CDT may be related to seed PCD. In the light of the crucial role played by ROS in PCD (Gechev et al. 2006), we then determined the localization of ROS production in longitudinal sections of embryos by CLSM with the probe DCFH-DA. The resulting fluorescent patterns within cells marked the sites of ROS formation (Fig 6). No detectable fluorescent signal was found in the embryos without DCFH-DA probe treatment and little fluorescence was found in control seeds (Fig. 6, I–VIII).
In an overview of the embryo (Fig. 6), fluorescence intensity was observed to gradually increase in the cells of embryos with deterioration (inset frames show more detail). In the images for 1 d of equilibration, only the cells near the epidermal layer of cortex had a strong ROS signal (Fig 6, g), and some scattered ROS signals were seen in the cells inside cotyledons (Fig 6, m). With longer CDT, the fluorescence intensity became stronger and reached a peak at 4 d of CDT (18% germination); the signal then became weaker by day 5 (Fig. 6, g–i). During equilibration, there was weaker fluorescence in the cotyledon, and thereafter, the ROS signal followed the same trend as in the cortex (Fig. 6, m–r). The first visible trace of ROS signal in the hypocotyl cells was seen at 2 d of CDT, and starting at 3 d, this was near the outer cells of hypocotyls. Although the ROS signal extended to the central part of the hypocotyls, the signal intensity was far lower than that seen in the cortex and cotyledon.
Next, we investigated the rate of ROS production in deteriorated seeds by monitoring H2O2 production. An immediate increase in H2O2 was detected during CDT. H2O2 peaked at 3 d of CDT, reaching 182% compared with control seeds and then decreasing. Similarly, seeds pretreated with AsA before aging exhibited decreased generation of H2O2. AsA effectively reduced H2O2 generation from 52 nmol g DW−1 to 32 µmol g DW−1 by the third day of CDT, which decreased slightly more by the fifth day of CDT (Fig. 7).
The activity of the antioxidant enzyme CAT did not significantly change during the first 3 d of CDT, but decreased after 4–5 d of CDT. The activities of other antioxidant enzymes, GR, SOD and APX, increased to day 2 of CDT by 253, 54.3 and 452%, respectively, and then decreased, although they generally remained above the activity levels seen in control seeds (Table 1). Very low amounts of AsA were detected in control seeds, and an accumulation of AsA and AsA/dehydroascorbate (DHA) was observed up to 2 d of CDT, increasing by 193 and 121%, respectively. Longer aging treatment decreased the AsA/DHA ratio and resulted in lower AsA content (Fig. 8A,B). The levels of reduced and oxidized glutathione (GSH and GSSG) varied in a similar way as those of AsA. At 2 d of CDT, GSH and GSH/GSSG increased by 26 and 63%, respectively, and thereafter, they decreased gradually with CDT to below the control level (Fig. 8C,D).
Table 1. Changes in the antioxidation enzyme activity of elm seeds during CDT
Ageing time (d)
CAT (µmol H2O2 mg−1 protein·min)
GR (µmol NADPH mg−1 protein·min)
SOD (U mg−1·protein)
APX (µmol AsA mg−1 protein·min)
The seeds were treated at 37 °C over water for CDT, and samples were taken after 0–5 d. The data are expressed as means ± SE from three experiments. Different letters indicate significant differences at P = 0:05 (least significant difference).
The seeds of many plant species will experience deterioration over time, although there are differences in longevity between species. Here, we provide reliable evidence that PCD occurs during elm seed CDT; as seen by DNA fragmentation, cyt c release and increase in DEVDase/caspase-3-like activity were checked during CDT. Our data indicate that ROS act as mediators in the apoptosis-like cell death process induced by CDT, which is similar to that reported in various types of PCD in both animals and plants (Danon et al. 2004; Gechev et al. 2006; Gao et al. 2008; Reape & McCabe 2008).
Elm trees are widely grown in northern China, and their seeds have high vigour, short longevity, and do not display dormancy. It was chosen as a suitable seed model for studying seed deterioration, because non-germination after CDT cannot be confused with dormancy, allowing a clear correlation of PCD occurrence and viability loss. Deteriorated seeds of elm showed considerable loss in viability, and the half-viability period (P50) was at 3 d (Fig. 1A). Under CDT, MC in equilibrated seeds attained a plateau at 1–3 d of CDT, and then showed a slow increase, but this was not statistically significant. This means 1 d is enough for seed equilibration at 37 °C over water (Fig. 1B).
The degree of electrolyte leakage during cell death is an important characteristic to distinguish PCD from cell necrosis (Pinto et al. 2012). Figure 1B showed that electrolyte leakage in seeds was a little higher but still not visible during the first 3 d compared with the control. However, it clearly increased after CDT for 4–5 d, and this indicated that the membrane system was considerably destroyed. A previous study on sunflower seeds has shown that the relationship between leakage and loss of seed viability cannot always be assumed, because no increase in electrolyte efflux occurred during CDT at 76 or 100% RH at 45 °C (Gidrol et al. 1989; Bailly et al. 1996). Lack of such a relationship between longevity and electrolyte leakage was also found in wheat (Lehner et al. 2008). A further study on sunflower seeds indicated the mechanism of loss of viability during deterioration depends on the seed MC (Kibinza et al. 2006). With a lower MC threshold value, H2O2 accumulated rapidly, but without causing lipid peroxidation; on the contrary, when seed MC was higher than this value, the loss of vitality was clearly associated with lipid peroxidation. This result implies the plasticity of the mechanisms involved in seed loss of viability.
We found that cellular morphology was dramatically altered during CDT, and this effect became more pronounced with time, where the temporal nature of the response was clear (Fig. 1C). TUNEL-positive nuclei were visible in the root cap and epidermis of the cotyledon on the first day of CDT, and with longer CDT, TUNEL-positive nuclei began to occupy the cotyledons, hypocotyls and shoot apices in sequence (Fig. 2). This observation suggested that the initiation of PCD in deteriorated seeds was asynchronous, and PCD occurred at specific sites and specific times during CDT. Our results are similar to previous reports examining embryogenesis in maize, and nucellus cell degeneration in Sechium edule Sw. seed (Giuliani et al. 2002; Lombardi et al. 2007).
There were no apparent differences in germination between the first day of CDT and control. Although TUNEL positivity occurred in 90% of epidermis cells of cotyledons and in 70% of root cap cells after 1 d of CDT, the seeds were healthy and fresh at these stages. Therefore, the initiation of nuclear DNA fragmentation as visualized by TUNEL may not exert a measurable impact on cell function. We noticed that cell nuclei were degraded in most mesophyll cells of cotyledons after 2 d of CDT. It is noteworthy that the effect of cotyledon cell death on seed germination was not clear, but it severely affected root growth, ultimately reduced seed vigour (Figs 1 & 2). Cell degradation is an active procedure that is important for plant survival and adaptation to unfavourable environmental conditions (Overmyer et al. 2005; Andronis & Roubelakis-Angelakis 2010). The elimination of damaged cells by PCD may be part of the resistance response, provided that sufficient cells remain viable to allow survival; however, if too many cells die, especially in the embryo, the entire seed will die. Therefore, PCD is vital to a seed in the resistance phase, and has been observed during seed deterioration before the loss of viability (Kranner et al. 2006, 2010).
DNA laddering is widely present in plant PCD, including accelerated ageing in pea (Kranner et al. 2006, 2010) and in sunflower (El-Maarouf-Bouteau et al. 2011). We were able to detect the presence of a typical DNA laddering with an increment of 160 bp in elm seed as of 2 d of CDT (89% germination), and then, strength increased until 5 d of CDT (Fig. 4). These results indicate that DNA laddering formation is a common feature associated with seed deterioration, at least in artificial ageing.
The release of cyt c from mitochondria to the cytosol plays an important role in initiating the caspase cascade of the mammalian and plant intrinsic PCD pathway (Garrido et al. 2006; Vacca et al. 2006; Giannattasio et al. 2008), such as in heat stress in cucumber (Cucumis sativus) (Balk et al. 1999) and in salinity-induced cell death of tobacco (N. tabacum) (Andronis & Roubelakis-Angelakis 2010). Consistent with these results, our results showed that the release of cyt c to the cytosol occurred early during CDT and in a time-dependent fashion (Fig. 5A). This release of cyt c was inhibited by the addition of AsA, indicating that cyt c release is related to the ROS production, which itself occurs soon after the induction of PCD (Fig. 6). Similar results were reported by Vacca et al. (2006), who showed that cyt c release was dependent on ROS production, as release did not occur in the presence of ROS scavengers. In animal systems, caspases are activated during the PCD execution phase. The existence of caspases in plants remains controversial (Sanmartín et al. 2005); however, caspase-3-like/DEVDase proteases have been shown to be activated during heat-induced PCD in tobacco cells (Vacca et al. 2006; Qu et al. 2009). Kranner et al. (2006) report the treatment of pea seeds with the caspase inhibitor zVAD-fmk prior to deterioration recovered viability by up to 20%, supporting the view that caspase-like proteins are involved in cell death during deterioration. In line with this result, we found the activation of caspase-3-like/DEVDase protease increased during CDT, further verifying that activity of caspase-3- like/DEVDase was reduced and seed germination improved when seeds were pretreated with the caspase-3-specific inhibitor Ac-DEVD-CHO (Fig. 5C,D). It is noteworthy that cyt c release appeared after the caspase activity reached an optimum level (Fig. 5A,B). There are two possible reasons for this occurrence. One is that cyt c release from the mitochondria into the cytoplasm may not be a hallmark of PCD but a conservative regulatory mechanism. Mitochondrial retrograde regulation (mitochondria to nucleus, MRR) is increasingly noted as an important regulatory/response mechanism for plants, animals and fungi. Protein released from the mitochondria in the regulation of protein expression is a normal adjustment mechanism and does not lead to cell death (Rhoads & Subbaiah 2007; Krause & Krupinska 2009). A previous study showed that the release of cyt c is not involved in an early signalling cascade during Petunia senescence (Xu & Hanson 2000). Another showed that caspase-like activities were detected in plants using mostly synthetic tetrapeptide substrates designed using the preferred cleavage site consensus of members of the mammalian caspase family. These substrates are not truly ‘caspase-specific’ and have overlapping specificities with various caspases and other proteases. It is therefore difficult to interpret caspase-like activity profiles in whole plant extracts. The detection of caspase-like activities, which correlates with PCD induction, does not demonstrate in itself an involvement in the process. Proof that caspase-like activities are required for completion of PCD must be obtained using a caspase inhibitor corresponding to the substrate used (Bonneau et al. 2008).
It must be emphasized that this process is unlikely to be the sole event involved in elm seed loss, though our results provide evidence that CDT induces PCD. TUNEL-negative nuclei were still observed in the tissues of the PC and shoot apex even after the germination capacity had decreased to 2% during late CDT (Fig. 2j,k). One possible explanation is that a mosaic of PCD and necrotic cells occurred in the CDT-exposed tissue. Mixtures of cells bearing signs of different modes of death within the same tissue have been described in studies of cell death in mammals (Levin et al. 1999), and have recently been proposed in plants (Greenberg & Yao 2004; Vuosku et al. 2009). Thus, signals emanating from the few cells undergoing necrotic cell death mediated by rampant oxidation via CDT-derived ROS may trigger PCD in the surrounding cells, resulting in large areas of affected tissue and lesion propagation.
ROS production in CDT-induced seed PCD
Oxidative damage has previously been correlated with seed degenerative processes and death, but how exactly this contributes to viability loss is unknown (Priestley 1986; Bailly et al. 1996; McDonald 1999; Kibinza et al. 2006). Kranner et al. (2006) proposed that the increase in the half-cell reduction potential of glutathione, presumably caused by ROS, may be part of a signalling cascade leading to PCD. H2O2 involvement has been reported in many types of PCD induced by abiotic stress, such as in heat shock (HS)-induced PCD in tobacco BY-2 cells (Vacca et al. 2004; Locato et al. 2008) and in hypoxia-induced lysigenous aerenchyma formation in Arabidopsis (Mühlenbock et al. 2007). Recently, the involvement of both ROS accumulation and NADPH oxidase gene induction had been reported in abscission zone (AZ) tissue PCD (Bar-Dror et al. 2011). Harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts, but mitochondrial ROS could be important in the orchestration of the cell suicide program (Garmier et al. 2007). To study the relation between ROS and PCD during CDT, in situ localization of ROS production was detected by the CLSM during CDT. Interestingly, the results showed that both were temporally and spatially consistent (Fig. 6). In addition, the rate of ROS production was also consistent with seed CDT (Fig. 7). The enhanced ROS signal was consistent with the increasing number of TUNEL-positive nuclei after CDT (Fig. 2), which was responsible for some of the physiological and cellular disturbances described in our study, including germination, electrolyte leakage and TUNEL detection (Figs 1 & 2). Our results clearly revealed that ROS preferentially accumulated in the root tip and cortical cells near the epidermis of embryos. Subsequent ROS signals began to accumulate gradually, expanding from the outer epidermis of cotyledons to the hypocotyl. This distinct spatial-temporal signature of ROS during CDT coincided with changes in DNA fragmentation as shown by TUNEL analysis (Figs 2 & 6), indicating that ROS generation is accompanied by cell death and PCD. Besides, cyt c release and increase of caspase-3-like/DEVDase activity, which occurred during CDT, could be suppressed by AsA, so an increase in ROS production was a prerequisite for CDT-induced PCD, which can be shown by the fact that seed germination improved if the ROS scavenger AsA was present during CDT. Recently, Kibinza et al. (2006) suggested that H2O2-induced ATP depletion could trigger cyt c release, which in turn could lead to PCD and loss of viability. Based on these experiments, we infer that ROS production was related to PCD process during CDT in elm seeds. ROS may have at least two roles in plant PCD: as a signalling molecule leading to opening of the permeability transition pore (PTP), which would lead to the release of cyt c, and generation of additional ROS, causing a feedback loop that would amplify the original PCD-inducing stress signal (Reape & McCabe 2008; Pinto et al. 2012).
The ROS level is tightly regulated by the balance between production and scavenging. The shift from a signalling to a deleterious role is related to the accumulation of ROS exceeding a threshold level, which leads to various cellular alterations and damage (Laloi, Apel & Danon 2004; Foyer & Noctor 2005; Bailly et al. 2008; Gadjev, Stone & Gechev 2008; Kranner et al. 2010). The early stages of CDT were characterized by marked increases in SOD, APX and GR activity, and in the GSH content (Table 1 & Fig. 8); it appears that the antioxidant system of most seed cells is prepared to protect against and repair in response to damage. However, this is insufficient to prevent H2O2 accumulation during subsequent CDT, so uncontrolled accumulation of ROS is likely to proceed continuously, leading to oxidative damage to a wide range of biomolecules and ultimately to necrosis and cell death (Figs 2 & 6). Kranner et al. (2006) proposed that the increase in the half-cell reduction potential of glutathione, presumably caused by ROS, may be part of a signalling cascade leading to PCD. Bailly et al. (1998) demonstrated that priming could reverse age-related damage to sunflower seeds, mainly by enhancing the activities of antioxidant enzymes, and therefore allowing the cells to escape oxidative stress.
In conclusion, the results of our morphological and biochemical analyses clearly showed that PCD may occur in the seed during CDT. We provided evidence that H2O2 accumulation during CDT was associated with PCD. Further research is required to establish the relationship between the occurrence of PCD and the underlying regulatory mechanisms during CDT.