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Keywords:

  • aleurone;
  • programmed cell death;
  • Hordeum vulgare;
  • gibberellic acid;
  • abscisic acid;
  • reactive oxygen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Summary

The cereal aleurone is widely used as a model system to study hormonal signalling. Abscisic acid (ABA) and gibberellins (GAs) elicit distinct responses in aleurone cells, ranging from those occurring within minutes of hormone addition to those that require several hours or days to complete. Programmed cell death is an example of a response in aleurone layers that is hormonally regulated. GAs promote cell death and cells in intact aleurone layers begin to die 24 h after GA treatment, whereas cell death of aleurone protoplasts begins 4 d after GA treatment. ABA prevents aleurone cell death and addition of ABA to cells pretreated with GA can delay cell death. Aleurone cells do not follow the apoptotic route of programmed cell death. Cells treated with GA, but not ABA, develop large, acidic vacuoles containing a spectrum of hydrolases typical of lytic compartments. Enzymes that metabolize reactive oxygen species are also present in aleurone cells, but ascorbate peroxidase, catalase and superoxide dismutase become less abundant after treatment with GA; activity of these enzymes increases or remains unchanged in ABA-treated cells. We propose a model whereby reactive oxygen species accumulate in GA-treated cells and lead to peroxidation of membrane lipids and plasma membrane rupture.

Abbreviations

RO, reactive oxygen species; HR, hypersensitive response; PSV, protein storage vacuole; PCD, programmed cell death; CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

The endosperm of barley, rice, wheat, oat and rye grain consists of two differentiated cell types. In the mature ripe grain, an outer layer of living aleurone cells envelopes the dead cells of the starchy endosperm (Olsen et al., 1995; Bethke et al., 2000). These dead starchy endosperm cells store carbohydrate and protein reserves that are utilized by the growing seedling after germination (Fincher, 1989). Cell death in the cereal starchy endosperm is unlike most other instances of programmed cell death (PCD) in that the contents of dead cells are not removed immediately after death.

The aleurone layer is a digestive gland that secretes the enzymes that break down the reserves stored in the starchy endosperm (Bethke et al., 2000). These enzymes digest the contents and the walls of starchy endosperm cells and leave an empty cavity. The carbon and nitrogen that are made available as a result of hydrolytic enzyme activity are used by the embryo to support the early stages of growth. When the aleurone layer has completed its digestive function it dies. Death of the aleurone cell is characterized as a form of PCD because it is tightly regulated by gibberellins (GAs) and abscisic acid (ABA) (Kuo et al., 1996; Bethke et al., 1999). PCD of wall-less aleurone protoplasts and isolated aleurone layers is stimulated by GA and arrested by ABA (Jones & Price, 1970; Bethke et al., 1999). Although there have been few recent studies of aleurone PCD in intact germinating cereal grains, the early work of Haberlandt (Haberlandt, 1884) with germinating rye indicates that PCD occurs in the aleurone during the normal course of germination.

PCD in plants takes many forms (Pennell & Lamb, 1997). As in animals, PCD in plants plays an important role in development whereby unwanted cells are removed, as in the case of suspensor cells during early embryogeny. Modelling of the mature plant body also requires removal of specific cells or groups of cells, and an example of this use of PCD occurs during tracheary element differentiation. This process has been investigated intensively over the last decade using the transdifferentiation of Zinnia elegans leaf mesophyll cells into tracheary elements (Fukuda, 1997). Extensive tissue remodelling also occurs in response to abiotic and biotic stresses in plants. Aerenchyma formation in maize roots during hypoxia is a result of PCD in spatially defined areas of the root cortex (He et al., 1994). Perhaps the best understood response of plant cells to pathogens is the hypersensitive response (HR), a kind of PCD that is elicited upon infection with avirulent pathogens (Hammond-Kosack & Jones, 1996).

Organ senescence is another example of PCD in plants, and cell death in the aleurone may represent a specialized form of senescence. Senescence allows the parent plant to scavenge mineral elements from dispensable or damaged organs before the organ is shed (Bleecker & Patterson, 1997). During leaf senescence, for example, almost all leaf nitrogen, phosphorus and potassium are redirected to rapidly growing parts of the plant (Gan & Amasino, 1997). During germination and seedling establishment, the aleurone layer mobilizes stored nutrients and exports them for the benefit of the embryo. Afterward it is a dispensable tissue and dies.

Molecules as diverse as ABA (Wang et al., 1996; Bethke et al., 1999; Young & Gallie, 2000), GA (Kuo et al., 1996; Bethke et al., 1999), ethylene (Grbic & Bleecker, 1995; Young et al., 1997), cytokinin (Gan & Amasino, 1995; Zavaleta-Mancera et al., 1999), fungal elicitors (Hammond-Kosack & Jones, 1996), reactive oxygen (Levine et al., 1994; Wojtaszek, 1997), nitric oxide (Delledonne et al., 1998; Leshem et al., 1998) and salicylic acid (Dempsey et al., 1999) have been proposed to be regulators of PCD. Whether any of these molecules interact directly with signalling pathways that lead to cell death is controversial. A role for reactive oxygen species (RO) as signalling molecules in PCD has been proposed by several groups working with the incompatible response of plants to pathogens (Lamb & Dixon, 1997). The production of superoxide (O2) at the plasma membrane by an NAD(P)H oxidase is one of the earliest responses observed following pathogen infection or elicitor stimulation (Hammond-Kosack & Jones, 1996). RO produced during pathogen attack may act as a trigger for the HR response and associated PCD. O2 is rapidly converted to H2O2, and other kinds of RO may also be produced (Hammond-Kosack & Jones, 1996). H2O2 may act directly to kill invading pathogens or may prevent their entry into tissues by stimulating lignification. Alternatively, because H2O2 can move rapidly across membranes, it may signal the presence of pathogens to cells remote from the site of pathogen entry. A role for nitric oxide (NO) in pathogen resistance and PCD is less well established. NO has a multiplicity of effects on plant growth and development, but its role as a signalling molecule has yet to be established (Wojtaszek, 2000).

In this manuscript we review the results of experiments designed to increase our understanding of PCD in cereal aleurone cells. We describe experiments with ABA and GA that indicate that cell death in this tissue is a form of PCD. We also show that PCD in the aleurone is different from apoptosis. Our data point to a key role for RO in PCD of aleurone cells and we describe experiments that link the cell’s ability to metabolize RO to cell viability.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Preparation of barley aleurone layers and protoplasts

Aleurone layers and protoplasts were prepared from deembryonated barley grains (Hordeum vulgare cv. Himalaya, 1991 and 1996 harvests, Washington State University, Pullman, WA, USA) as described previously (Bethke et al., 1999; Fath et al., 1999). Aleurone protoplasts were either incubated in 25 µM GA and 5 µM ABA (GA-treated) or 5 µM ABA (ABA-treated) for the time indicated. In experiments that used ABA to reverse the effect of GA on cell death, we added a fivefold excess of ABA (125 µM) to GA-containing (25 µM) media. Protoplasts used for DNA quantification were prepared using the method of Lin et al. (1996). This method incorporates an extensive wash step that utilizes repeated centrifugation on Percoll (Sigma, St. Louis, MO, USA) density gradients to remove nucleases found in the Onozuka cellulase (Yakult Pharmaceutical Ind. Co., Tokyo, Japan) from contaminating the DNA extract and degrading the DNA during the isolation procedure. For DNA isolation, only living protoplasts were used. Living protoplasts were separated from dead protoplasts by Nycodenz (Sigma) density centrifugation as described (Bethke et al., 1996). α-Amylase activity was measured using the starch-iodine method.

Determination of cell viability

Viability of protoplasts was determined by counting the number of live cells in a sample using an improved Neubauer haemocytometer (Fisher Scientific, Pittsburh, PA, USA). Live protoplasts appeared round and had an intact PM. The PM of protoplasts becomes ruffled immediately after death, and the cell slowly collapses to form a compact mass of cellular debris that is easily distinguished from live cells. Protoplasts were examined using a Zeiss Axiophot microscope (Zeiss, Thornwood, NJ, USA) equipped with a Plan-Neofluor 40× dry objective, 0.75 numerical aperture objective and differential interference contrast optics. Protoplasts were photographed with Kodak Ektachrome 160T film (Eastman Kodak, Rochester NY, USA).

Viability of cells in intact aleurone layers was determined by staining living aleurone layers with fluorescein diacetate (FDA, 2 µg ml−1 in 20 mM CaCl2, Molecular Probes, Eugene, OR, USA) for 15 min followed by N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)-hexatrienyl) pyridinium dibromide (FM 4–64, 20 µM in 20 mM CaCl2, Molecular Probes) for 3 min. Layers were observed with a Zeiss Axiophot microscope (Zeiss) and images where captured using Ektachrome 160T film (Eastman Kodak). Living, green fluorescing cells and dead, orange-red fluorescing cells were counted.

H2O2-Induced Death

Washed protoplasts (5 µl) were added to 2 ml of baseline culture medium containing 3.25 mM (0.01%) or 325 mM (1%) H2O2. The sample of protoplasts was scanned at 20 min (1% H2O2) or 60 min (0.01% H2O2) intervals and individual protoplasts scored as either live or dead. Protoplasts were illuminated with incandescent light only while measurements were made. Protoplasts that appeared turgid and spherical were judged alive. Aspherical protoplasts and those with a ruffled PM were scored dead.

In vivo H2O2 measurements

Fluorescent probes were purchased from Molecular Probes and an aliquot of stock dye solution was added to the medium in which protoplasts were cultured. For in vivo H2O2 measurements washed protoplasts were incubated in 25 µM 6-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate di(acetoxymethly ester) (CDCDHFDA-AM) for 2 h, then washed four times with baseline culture medium. Protoplasts were transferred to wells on microscope slides and illuminated using the mercury light source on a Zeiss Axiophot microscope fitted with a 20× Neofluor objective and optical filters (495 nm excitation, 505 nm dichroic mirror, 535 + 25 nm emission; Chroma Technology Corp., Brattleboro, VT, USA) that allowed both blue-light illumination and visualization of the oxidized fluorescent probe. Illumination was controlled by cycling between the open and closed positions of a filter wheel (Sutter Instrument Co, Navato, CA, USA). Quantitative images were captured using IPLab 3.1 software (Signal Analytics Corp., Vienna, VA, USA) and a cooled, charged-coupled device camera (Princeton Instruments, Trenton, NJ, USA) with a 150 ms exposure. Data analysis was performed with IPLab software. When H2O2 was added to CDCDHFDA-AM loaded protoplasts, 15 µl 6.5 mM (0.02%) H2O2 in baseline culture medium was added to 15 µl washed protoplasts. Samples were illuminated only when data were recorded.

For quantification of H2O2 using dihydrofluorescein diacetate (DHFDA, Molecular Probes), washed protoplasts were added 1 : 5 to baseline culture medium containing 10 µM DHFDA. Without additional washes, protoplasts were transferred to wells on microscope slides and images captured as described above. Imaging began 5 min after protoplasts were added to DHFDA.

DNA isolation and electrophoresis

DNA from barley aleurone protoplasts and layers was purified using a modified method described by Wang et al. (1996). Briefly, frozen protoplasts were thawed on ice in the presence of lysis buffer (750 µl 0.1 M Tris pH 7.5, 50 mM EDTA, 0.5 M NaCl and 100 µl 10% SDS) and vortexed. Without further incubation in lysis buffer, NaAc was added to a final concentration of 0.3 M, mixed and incubated for 10 min on ice. After centrifugation for 1 min at c. 12  000 g, the supernatant was transferred to a new tube. One volume of 100% isopropanol was added, mixed by inversion, incubated on ice for 1 min and centrifuged for 5 min at c. 12  000 g. The supernatant was discarded and the pellet was dissolved in 250 µl TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) and 250 µl CTAB buffer (0.2 M Tris pH 7.5, 50 mM EDTA, 2 M NaCl and 2% CTAB [etyltrimethylammonium bromide; Aldrich, Milwaukee, WI, USA]) and incubated for 15 min at 65°C. The DNA was extracted with 1 volume of chloroform and centrifuged. The aqueous phase was transferred to a new tube and two volumes of 100% EtOH were added. DNA was allowed to precipitate for at least 20 min at −20°C. The DNA was washed, dried and resuspended in 50 µl TE buffer supplemented with 1 mg ml−1 RNaseA. DNA was isolated from aleurone layers in a similar manner except that 15 layers were ground to a fine powder in liquid N2. DNA was quantified by measuring the absorbance at 260 and 280 nm. The quality of the DNA was examined by loading 4 µg of DNA on a 1.5% agarose gel followed by staining with 0.5 µg ml−1 ([w/v], final concentration) ethidium bromide.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Is cell death in the cereal aleurone programmed?

Cell death in the cereal aleurone layer is promoted by GA and can be blocked by ABA (Fig. 1a; Bethke et al., 1999; Fath et al., 2000). We interpret these observations as indicating that death of cereal aleurone cells is a form of PCD. Aleurone cells begin to die about the same time that the GA-induced secretion of acid hydrolases, mainly α-amylase, has ceased (Fig. 1b). In one typical experiment the number of living protoplasts was reduced by 40% between 4 and 6 d after GA-treatment and all but 10–30% of the protoplast population died after incubation in GA for 2 additional days (Fig. 1b). On the other hand, aleurone protoplasts or cells in intact aleurone layers do not secret α-amylase and remain viable for extended periods when incubated in ABA (Fig. 1a,b; Bethke et al., 1999; Bethke & Jones, 2001).

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Figure 1. Hormonal regulation of cell death in barely aleurone protoplasts. (a) Number of living protoplasts per flask after treatment of freshly isolated protoplasts with 5 µM abscisic acid (ABA) for the time indicated (closed squares). The number of living gibberellin(GA)-treated protoplasts from Figure 1(b) is plotted as a dashed line for comparison. (b) Cell death (closed circles) and α-amylase secretion (open circles) from GA-treated aleurone protoplasts. Cell death was quantified by counting the number of living cells at each time point using a haemocytometer. α-Amylase activity was measured as accumulated activity in the incubation medium. Each time point represents the mean of three flasks ± standard deviation of the mean.

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Support for the idea that aleurone cell death is a form of PCD also comes from experiments showing that ABA and GA act antagonistically to tightly regulate viability and death in these cells. Addition of GA to protoplasts incubated in ABA for up to several weeks brings about the synthesis and secretion of α-amylase and subsequently cell death (Bethke et al., 1999).

Perhaps the most convincing evidence that ABA is an important regulator of PCD in barley aleurone comes from experiments showing that the synthesis and secretion of α-amylase can be uncoupled from cell death by ABA (Fig. 2). If ABA is added to protoplasts preincubated in GA for 24 h and incubation is continued for a further 5–7 d, production of α-amylase is unaffected by the added ABA relative to controls incubated in GA alone (Fig. 2a). The onset of cell death, however, is significantly delayed (Fig. 2b). Thus, by 8 d of incubation in GA alone more than 80% of protoplasts died, but when ABA was added at 24 h to GA treated cells fewer than 30% of cell had died (Fig. 2b). These data also exclude the possibility that cell death results from exhaustion of nutrients as a consequence of hydrolase production, specifically α-amylase.

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Figure 2. Abscisic acid (ABA) delays PCD in gibberellin(GA)-treated aleurone protoplasts. α-Amylase secretion (a) and cell death (b) of aleurone protoplasts treated with 25 µM GA for the number of days indicated with (open squares) or without the addition of 125 µM ABA at 24 h (closed circles). Each point represents the mean of four flasks and each flask was sampled only once.

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Secreted proteases have been implicated in PCD of zinnia tracheary elements (Groover & Jones, 1999). We have shown that death of GA-treated aleurone protoplasts is unlikely to result from the accumulation of toxic amounts of secreted hydrolases including proteases and nucleases in the incubation medium. Incubation of freshly prepared barley aleurone protoplasts in medium (conditioned medium) in which GA-treated protoplasts were incubated for 8 d, and where more than 90% of the cells had died, did not bring about cell death. Although conditioned medium contained secreted hydrolases as well as vacuolar hydrolases that would have been released as cells died, death of freshly prepared protoplasts was not accelerated relative to protoplasts incubated in fresh, hydrolase-free medium (Bethke et al., 1999).

Aleurone cells develop lytic vacuoles after exposure to GA

Aleurone cells contain many protein storage vacuoles (PSV, Fig. 3a) and these store phytate and carbohydrate in addition to storage proteins (Bethke et al., 1998). After aleurone cells perceive GA the contents of the PSV are hydrolysed and these organelles fuse, eventually forming one large vacuole (Fig. 3b,c). The pH of PSV in GA-treated cells is 5.5 or less, and these vacuoles contain proteases and other hydrolytic enzymes (Bethke et al., 1996; Swanson & Jones, 1996). The vacuoles in cells exposed to ABA, on the other hand, have a pH of approx. 7 and lack most of the hydrolase activities found in GA-treated cells. Using isolated, intact vacuoles, we showed that PSVs from GA-treated aleurone protoplasts contain several cysteine and aspartic protease activities whereas PSV from dry layers and ABA-treated protoplasts have fewer protease activites (Bethke et al., 1996). When compared to the protease activity in the cytosol and other organelles, we showed that most of the acidic protease activity in the aleurone cell is in the vacuole.

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Figure 3. Barley aleurone cells become highly vacuolate before gibberellin(GA)-induced programmed cell death (PCD) and PCD is accompanied by a rapid loss of plasma membrane integrity. Zero day shows the organization of organelles in a freshly isolated protoplast (a). Protoplasts incubated in GA for 2 d (b) and 4 d (c) show progressive vacuolation of the cell. A dead cell is shown in (d,e). Note the development of the large protein storage vacuole (PSV) 4 d after GA-treatment (c) that arises from fusion of the smaller PSV seen in (a,b). Bar, 10 µm.

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We used antibodies to a previously characterized barley aspartic protease, phytepsin (Runeberg-Roos & Saarma, 1998), to confirm the identity of one activity in aleurone vacuoles. Using isolated, intact vacuoles we showed that phytepsin is localized to the lumen of the PSV in GA-treated aleurone cells (Bethke et al., 1996). PSV in GA-treated cells also contain phosphatase activities and the activities of these enzymes are lower in ABA-treated cells (Gabard & Jones, 1986).

We confirmed that PSV of aleurone cells contain active cysteine proteases in vivo by using a fluorogenic substrate (Swanson et al., 1998). When this substrate was loaded into intact living cells or into intact vacuoles isolated from living cells, cysteine protease activity could be demonstrated. These experiments establish that the cysteine protease activities in aleurone extracts are unlikely to be artefacts of enzyme isolation. One of the cysteine proteases induced by GA in the aleurone cell is aleurain (Holwerda & Rogers, 1992), a homologue of senescence-induced cysteine proteinase 2 in maize (Griffiths et al., 1997) and of γ-oryzain in rice (Watanabe et al., 1991). We designed specific primers for cysteine proteases and showed that homologues of α- and β-oryzains also occur in barley, and RNA blotting showed them to be expressed in the aleurone layer. The expression of α-oryzain, β-oryzain, and aleurain is up-regulated by GA and strongly down-regulated by ABA. Low amounts of α-oryzain, β-oryzain and aleurain mRNAs correlate with an absence of cysteine protease activity in PSV of ABA-treated protoplasts (R. Meza Romero, J. Zhang and R. Jones, unpublished).

Aleurain, the α- and β-oryzain homologues, and other nonsecreted hydrolases are likely to be important for aleurone cell death. Aleurain is homologous to the mammalian lysosomal enzyme cathepsin H (Rogers et al., 1985) and was localized to a distinct lysosome-like organelle in aleurone cells (Holwerda et al., 1990). We speculate that aleurain functions as a lysosomal enzyme in the aleurone cell, perhaps playing a role in organelle turnover as a prelude to cell death. Vacuolar hydrolases in the aleurone cell, as well as secreted hydrolases that accumulate in the endosperm cavity, may function to digest the remnants of the dead aleurone cell. In this regard the final stages of tracheary element formation in zinnia (Fukuda et al., 1998) may resemble those that occur in the cereal endosperm. In zinnia cells, vacuolar hydrolases are released into the cytoplasm and digest the contents of the cell.

Do aleurone cells die by apoptosis?

The hallmarks of apoptosis include chromatin condensation, blebbing of the nuclear membrane, cleavage of DNA at internucleosomal sites, and fragmentation of the cell into apoptotic bodies (Kerr & Harmon, 1991). Apoptotic bodies are engulfed by phagocytes, thus removing the remnants of the cell corpse, but this does not occur in plants. Furthermore, there is little evidence that apoptotic body formation occurs during plant PCD. For cereal aleurone cells, fragmentation of the cell does not occur before cell death, even in wall-less protoplasts (Fig. 3).

The formation of DNA fragments differing in length by approx. 180 bp has been used as a diagnostic feature of apoptosis, and the appearance of a 180-bp DNA ladder following electrophoresis is consistent with apoptotic PCD. When methods were used to ensure that DNA fragmentation did not occur following DNA isolation from barley aleurone protoplasts and layers, we found no evidence of internucleosomal cleavage of DNA (Fig. 4a). We could show, however, that the amount of DNA per living aleurone cell declined with prolonged incubation in GA but not ABA (Fig. 4b). This occurred after 4 d of incubation in GA, when the synthesis of hydrolases is at an end and cells have become highly vacuolate. This decline in DNA did not result in the loss of all nuclear DNA since dead aleurone cells contain an identifiable nucleus that stains with the TUNEL reagent, SYTO 11 or DAPI (Bethke et al., 1999; Fath et al., 1999).

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Figure 4. (a) Agarose gel electrophoresis of DNA (4 µg lane−1) isolated from barley aleurone protoplasts incubated in abscisic acid (ABA) or gibberellin (GA) for up to 5 d. A 500-bp DNA ladder was used as a molecular weight marker (Mr). (b) Number of living protoplasts (top) and amounts of DNA per live protoplast (bottom) following incubation in GA (closed circles) or ABA (closed squares).

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Barley aleurone cells contain at least three different nucleases whose activities are up-regulated with GA and are not present in ABA-treated cells (Fath et al., 1999). There is a strong correlation between increased nuclease activity, the decline in total DNA in living GA-treated aleurone cells, and cell death (Fath et al., 1999). Nuclease activity, DNA degradation and cell death could be prevented by the guanylyl cyclase inhibitor LY83583, making cGMP a likely component of the GA-signalling pathway leading to PCD (Bethke et al., 1999; Fath et al., 1999).

The execution of death

Time lapse photomicrography shows that GA-treated aleurone cells die after almost all of the cell’s volume is occupied by a large lytic vacuole (Fig. 3). The cytoplasm of GA-treated cells is confined to a narrow zone sandwiched between the PM and tonoplast, except in the region containing the nucleus. Other organelles such as mitochondria, the Golgi apparatus, and glyoxysomes accumulate near the nucleus (Lonsdale et al., 1999). As the cell vacuolates, organelles are lost, although the mechanism whereby organelles disappear is not known. The amount of mitochondrial protein is reduced by > 50% during the first 24 h of incubation of aleurone layers in GA, and light and electron microscopy indicate that other organelles are also reduced in number (A. Fath, P. Bethke, J. Lonsdale and R. Jones, unpublished). We have carried out detailed investigations using electron microscopy techniques such as high pressure freezing-freeze substitution that give excellent preservation of organelles (Lonsdale et al., 1999) but have yet to establish the mechanism of organelle turnover.

Aleurone cell death is stochastic and results from a loss of PM integrity. This causes turgor to be lost (Fig. 3d), and results in shrinkage of the cell corpse (Fig. 3e). Changes in the permeability of the PM are easy to follow in dying aleurone cells when vital stains such as the plasma-membrane impermeable DNA-binding fluorochrome YO-PRO-1 are included in the incubation medium (Bethke et al., 1999). With this approach we could show that morphological changes associated with death occur very rapidly, and are accompanied by staining of the nucleus with YO-PRO-1 (Bethke et al., 1999).

RO are key players in aleurone PCD and our data show that RO may be the cells’ executioner. Several lines of evidence suggest that RO kill aleurone cells that are programmed to die as a result of prolonged incubation in GA. A key experiment that lead to our investigation of the role of RO in aleurone cells was one that showed that bright blue or UV light accelerated death of GA-but not ABA-treated cells. Light-induced death of GA-treated protoplasts was manifest as a ruffling of the PM and a loss of PM integrity and was similar to that observed for GA-treated protoplasts kept in dim light (Bethke & Jones, 2001). An action spectrum of the light response suggests the involvement of a flavin-like photoreceptor with maximal activity at c. 465 nm and c. 380 nm. Using fluorescent probes that are sensitive to H2O2 we showed that blue or UV light increased the rate of H2O2 production in aleurone protoplasts (Bethke & Jones, 2001). Elevated H2O2 following exposure to light correlated with rapid cell death in GA-treated protoplasts (Bethke & Jones 2001). By contrast, ABA-treated cells did not die when illuminated with bright blue or UV light (Bethke & Jones, 2001). These experiments suggested that GA-treated cells were less able to withstand the increase in RO brought about by light when compared to their ABA-treated counterparts.

To test the hypothesis that GA-treated aleurone cells are more susceptible to RO than ABA-treated cells, we exposed aleurone protoplasts and layers to H2O2 after incubation in GA or ABA (Fig. 5). To carry out this experiment in intact aleurone layers, we monitored viability and cell death of aleurone cells by simultaneously staining living and dead cells with fluorescent probes (Fig. 5a–e). Fluorescein diacetate is taken up by living aleurone cells and fluoresces green whereas FM 4–64 accumulates rapidly in dead cells and fluoresces orange-red. When aleurone layers were preincubated in GA for 24 h, 80% of the cells in the layer were alive. Incubation of these cells in H2O2 for an hour resulted in the death of almost all cells (Fig. 5d,e), whereas incubation for 1 h without H2O2-treatment had no significant effect on cell viability (Fig. 5c,e). For layers treated with ABA for 24 h, virtually no cells died when exposed to H2O2 for 1 h (Fig. 5b,e). Similar experiments were carried out with protoplasts (Fig. 5f,g). Protoplasts incubated in GA were highly sensitive to H2O2 whereas protoplasts incubated in ABA were refractory to H2O2 and did not die (Fig. 5f,g).

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Figure 5. The effect of H2O2 on cell death in barley aleurone layers (a–e) or protoplasts (f,g). (a–d) Images of aleurone layers incubated in abscisic acid (ABA) (a,b) or gibberellin (GA) (c,d) for 24 h and then with (b,d) or without (a,c) exposure to 1% H2O2 for 1 h. Layers were stained with fluorescein diacetate and N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienyl) pyridinium dibromide and visualized by epifluorescence microscopy. Live cells appear green (FDA) and dead cells orange-red (FM 4–64). (e) Quantification of cell death following incubation in H2O2 by counting living and dead cells after staining of layers with FDA and FM 4–64. (f,g) Protoplasts were incubated in 25 µM GA (open circles) or 5 µM ABA (closed circles) for 7 d and exposed to 325 mM (1%) H2O2 (f) or 3.25 mM (0.01%) H2O2 (g) for the times indicated and the percentage of live cells was determined for each time.

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We investigated the biochemical basis for the resistance of ABA-treated protoplasts to H2O2 and showed that viability of ABA-treated cells correlated with high rates of activity of catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) (Fath et al., 2001). By contrast to this, the amounts and activities of these RO-metabolizing enzymes were reduced or not detectable after 24 h of incubation in GA (Fath et al., 2001). We hypothesize that aleurone cell viability is intimately connected to the presence of RO-metabolizing enzymes. When the activities of these enzymes decline, as in GA-treated cells, aleurone cells are unable to catabolize RO. We propose that RO are the executioners of death in the aleurone cell.

What is the source of RO in the barley aleurone cell? Aleurone cells are nonphotosynthetic and therefore do not generate RO as byproducts of photosynthesis. On the other hand, aleurone cells store large amounts of triglycerides in the oleosomes that occupy as much as 25% of the aleurone cell (Jones, 1969). Cells in the aleurone layer carry out gluconeogenesis (Doig et al., 1975) so that energy and metabolic building blocks can be made available for the synthesis and secretion of enzymes needed to digest the storage reserves in the starchy endosperm. As these triglycerides are converted to sugars, the reactions of β-oxidation generate RO. It is notable that the first enzyme of β-oxidation, acyl CoA oxidase, is a flavin-containing enzyme that produces one mole of H2O2 for each acetyl CoA released. This same enzyme may contribute to the production of RO when aleurone cells are illuminated with bright blue or UV light. Blue and UV light have been shown to stimulate H2O2 production in mammalian cells and it was postulated that the source of the H2O2 was a flavin-containing oxidase (Hockberger et al., 1999).

Our working model for GA-induced RO-mediated cell death in barely aleurone is presented in Fig. 6. H2O2 is produced at relatively high rates, in part by β-oxidation in the glyoxysome. H2O2 is highly mobile within the cell and when CAT and APX activities are high, it can be assumed that this H2O2 is metabolized rapidly. When the amount of RO-metabolizing enzymes declines in GA-treated aleurone cells, the intracellular concentration of RO increases, as does the potential for damage to cell membranes, DNA and proteins. We speculate that decreased mitochondrial activity may contribute to an increase in RO by slowing the rate of antioxidant regeneration along with reducing the rate of ATP production. Higher than normal concentrations of RO may lead to peroxidation of membrane lipids and cause a loss of membrane integrity that ultimately results in cell death.

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Figure 6. A model for gibberellin(GA)-induced RO-mediated cell death in barley aleurone. CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase; RO, reactive oxygen species; PM, plasma membrane.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the National Science Foundation (grant#IBN-9818047) and Torrey Mesa Research Institute to Rusell L. Jones. Figures 1 and 2a were taken with permission from Bethke et al. (1999), Figure 4 from Fath et al. (1999) and figure 5 from Bethke & Jones (2001).

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  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
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