Journal of Experimental Botany, Vol. 53, No. 372, pp. 1273-1282,
May 15, 2002
© 2002 Oxford University Press
Original Papers |
Active oxygen and cell death in cereal aleurone cells
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA
Received 10 July 2001; Accepted 29 October 2001
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Abstract |
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The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H2O2 accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H2O2 and are not killed. It was shown that light elevated intracellular H2O2, and that the rise in H2O2 was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H2O2 is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6–18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.
Key words: Abscisic acid, active oxygen, aleurone, cell death, gibberellic acid.
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Introduction |
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In this article, the roles of active oxygen species (AOS) in programmed cell death (PCD) in the cereal aleurone are explored. The cereal aleurone is a useful model system for these studies. The aleurone layer differentiates from starchy endosperm cells as early as 8–10 d after pollination (Bosnes et al., 1992
). The aleurone layer is a terminally differentiated, non-dividing tissue that is non-photosynthetic at functional maturity (Bethke et al., 2000). It functions as the principal store of cations and phosphate in the grain (Lott et al., 2000) and as a digestive gland that synthesizes and secretes a range of acid hydrolases (Fincher, 1989). By the time the grain is mature, all of the starchy endosperm cells have died, but the aleurone layer remains viable. After germination and during early seedling growth the starchy endosperm and aleurone layer are depleted of their reserves. For the aleurone layer, this process begins with the export of minerals and the secretion of hydrolases and ends when the remnants of the cells are digested by apoplastic enzymes. In this regard, aleurone cell death resembles leaf senescence. Details of endosperm mobilization and aleurone cell death were first described in the nineteenth century by Haberlandt (Haberlandt, 1884).
One advantage of the mature cereal aleurone layer as an experimental system for studies of AOS and PCD is that it is devoid of chloroplasts. AOS are produced during photosynthesis, and chloroplasts have evolved a variety of mechanisms to protect against the deleterious effects of AOS (Noctor and Foyer, 1998; Asada, 1999; Niyogi, 1999). Indeed, more is known about the sources of AOS in the chloroplast and their roles in photo-oxidative damage than about other aspects of AOS in plants. AOS produced during photosynthesis, however, may mask an AOS signal, or AOS produced specifically as part of a cell death programme. Aleurone cells lack photosynthetic pigments and electron microscopy shows that plastids in the aleurone cells of barley are few in number and have poorly developed internal membranes (Jones, 1969b). The lack of a functional photosynthetic apparatus in the aleurone cells excludes photosynthesis as a source of AOS in this cell.
Because AOS have been implicated in cell death responses in plants there has been renewed interest in understanding how and where AOS are produced in plants. AOS are implicated in cell death resulting from pathogen attack (Heath, 2000), senescence (Rubinstein, 2000), elevated atmospheric ozone (Rao et al., 2000), blue and UV light (Bethke and Jones, 2001), and in lesion mimic mutants (Mittler and Rizhsky, 2000). Two roles in plant PCD have been proposed for AOS. First, they can act as signalling molecules to initiate a response cascade that results in death (Lamb and Dixon, 1997). Alternatively, they can have a direct role in killing plant cells (Bethke and Jones, 2001). These roles of AOS are not mutually exclusive. Evidence that AOS are the agents that kill cells under a variety of developmental, environmental and pathological conditions comes from experiments in which AOS kill cells quickly and modulation of AOS production protects against the damaging effects of AOS. For example, antisense suppression of ascorbate peroxidase (APX) and catalase (CAT), two enzymes that are known to detoxify AOS in plants, makes tobacco hypersensitive to pathogen infection (Mittler et al., 1999). On the other hand, there is evidence that AOS are necessary but not sufficient to cause PCD in the case of death brought about by ozone (Rao et al., 2000). And in elicitin-treated suspension-cultured cells, AOS produced by the DPI-sensitive oxidase were neither necessary nor sufficient for PCD (Dorey et al., 1999; Sasabe et al., 2000).
Among the questions that remain to be resolved about AOS in plants, especially in the context of cell death, are the identity of the AOS species that participate in PCD and their sites of production within the cell. In animal cells, mitochondria are the principal source of AOS. In the mitochondria of mammals, the superoxide anion (
) is generated as electrons are transported to O2 via cytochrome oxidase. Superoxide is vectorially transported into the mitochondrial matrix where a manganese-dependent superoxide dismutase (Mn-SOD) catalyses the conversion of to H2O2 (reviewed in Cadenas and Davies, 2000). Mitochondria also contain catalases that convert H2O2 to O2 and H2O. Calculations of the steady-state concentrations of and H2O2 in mitochondria from rat heart and liver show that the amount of H2O2 is 25–70 times that of (Cadenas and Davies, 2000). Membranes are freely permeable to H2O2, but much less so to (Thannickal and Fanburg, 2000). Thus, H2O2 produced by mitochondria is free to diffuse into the cytosol, but is retained within the mitochondria.
The electron transport chain in plant mitochondria differs from that in mammals in that plant mitochondria use a branched electron transport system. One branch is a conventional pathway that uses cytochrome c to couple the reduction of O2 to ATP production by the mitochondrial ATP synthase (Siedow and Day, 2000). The second branch for electron transport to O2 is via a rotenone-insensitive NADH/NADPH dehydrogenase and an alternative oxidase (AOX). The second pathway bypasses those steps that generate the proton motive force required for ATP production and does not produce . By manipulating electron transport in isolated mitochondria (Purvis, 1997) or by antisense suppression of AOX in intact plants (Maxwell et al., 1999), it has been shown that less is produced by plant mitochondria when the AOX pathway is operating. Based on these data it has been proposed that, by directing electrons to the AOX pathway, plants limit the overreduction of the quinine pool and thus dramatically reduce the production of AOS (Vanlerberghe and McIntosh, 1997).
Mitochondria in mammals have another enzyme system, monoamine oxidase, that is capable of producing AOS (Cadenas and Davies, 2000; Thannickal and Fanburg, 2000). Monoamine oxidase is located in the outer mitochondrial membrane and catalyses the two-electron reduction of O2 to H2O2. This enzyme system catalyses the oxidative deamination of primary aromatic amines, long-chain diamines and tertiary cyclic amines. In mammalian cells this enzyme can generate large amounts of H2O2 (Cadenas and Davies, 2000). There is as yet no evidence that plant mitochondria contain this enzyme system.
Peroxisomes and glyoxysomes are also major sources of AOS. In photosynthetic cells, peroxisomes generate H2O2 directly via the two electron reduction of O2 by the flavoprotein glycollate oxidase (Corpas et al., 1993; del Rio et al., 1998). In cells metabolizing fatty acids, glyoxysomes generate AOS via fatty acyl CoA oxidase. Fatty acyl CoA oxidase is also a flavoprotein that reduces O2 directly to H2O2, and glyoxysomes, like peroxisomes, contain high concentrations of catalase (Beevers, 1979). Because membranes are readily permeable to H2O2, however, the H2O2 produced in both of these organelles is likely to influence cytosolic AOS concentrations (Pastori and del Rio, 1997). AOS production in senescing plant tissues is thought to involve the peroxisome/glyoxysome (del Rio et al., 1998) and this organelle may be a site of AOS production during stress responses in plants.
In plants, the plasma membrane (PM) NAD/NADP oxidase is another class of enzyme that is a significant source of AOS (Rubinstein and Luster, 1993; Lamb and Dixon, 1997; Murphy et al., 1998). This enzyme is implicated in PCD during the hypersensitive response. Like the NADPH oxidase located in the PM of phagocytes and other mammalian cells (reviewed in Thannickal and Fanburg, 2000), the plant enzyme is inhibited by diphenyliodonium (DPI) and several groups have shown that synthesis of AOS at the PM is responsible for the burst in AOS that occurs on elicitation or infection of plant cells with pathogens (Low and Merida, 1996; Lamb and Dixon, 1997). The enzymes that generate AOS at the PM of plants have yet to be fully characterized, but in cultured rose cells, a superoxide synthase (SOS) has been described (Murphy et al., 1998). The SOS of rose cells uses NADH and NADPH as substrates, requires flavins for activity and are inhibited by DPI (Murphy and Auh, 1996).
The endomembrane system of eukaryotic cells also contains a variety of oxidases that have the potential to generate AOS (Thannickal and Fanburg, 2000). Cytochrome P450 mono-oxygenases may be especially important in this regard (Halkier, 1996). At least 400 cyt-P450 genes are known in Arabidopsis (Ohkawa et al., 1999) and these play diverse roles in plant metabolism including the regulation of hormone biosynthesis, the synthesis of phenylpropanoids, alkaloids and terpenoids, the oxidation of fatty acids, and the detoxification of xenobiotics (Chapple, 1998). Although the cyt-P450 enzymes can produce and/or H2O2, little is known about the potential roles of these AOS in cell signalling or PCD.
Cereal aleurone cells store large amounts of neutral lipid in specialized oleosomes (Jones, 1969b), and as much as 25–30% of aleurone cell volume is occupied by oleosomes (Jones, 1969a). Barley (Jones, 1972; Holtman et al., 1994) and wheat (Doig et al., 1975) aleurone cells contain catalase and the enzymes of the glyoxylate cycle, indicating that lipid breakdown and gluconeogenesis are occurring. Although the presence of large reserves of neutral lipid in aleurone cells may at first glance appear anomalous, it is likely that aleurone cells cannot access the abundant reserves of carbohydrate in the starchy endosperm until secreted hydrolases such as 
-amylase are synthesized. There is speculation that the early steps in reserve mobilization by the cereal aleurone layer are dependent on sugars produced via ß-oxidation and the glyoxylate cycle. A by-product of these reactions is the synthesis of H2O2. In this context it is interesting to note that lipoxygenase is the most abundant enzyme in the oleosome of germinating soybean seeds (Feussner and Kindl, 1992; Feussner et al., 1995a) and it has been proposed that lipoxygenase plays a key role in neutral lipid degradation (Feussner et al., 1995b). Lipoxygenase has also been shown to produce AOS in plants.
In this article, the roles of AOS in PCD in barley aleurone are examined. Experiments are described where PCD is monitored as AOS concentrations are experimentally manipulated. Experiments are also described with gibberellic acid (GA) and abscisic acid (ABA) that show that cell death and AOS concentrations are strongly positively correlated with the amounts of enzymes that metabolize AOS. The sources of AOS in barley aleurone cells are discussed with emphasis on the roles of glyoxysomes and mitochondria.
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Materials and methods |
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Plant material: preparation of barley aleurone layers and protoplasts
Barley (Hordeum vulgare L. cv. Himalaya) aleurone layers and aleurone protoplasts were isolated from grain of the 1991 harvest obtained from the Department of Agronomy, Washington State University, Pullman, WA, USA. Aleurone layers were prepared as described previously (Fath et al., 1999
) and incubated in media containing 20 mM CaCl2 and 5 µM of ABA or GA. Protoplasts were prepared from barley grain as described previously (Bethke et al., 1999). Freshly prepared protoplasts incubated in baseline culture medium containing Gamborg's salts, 20 mM CaCl2 and 5 µM ABA are referred to as ABA-treated. Protoplasts incubated in baseline culture medium containing 25 µM GA are referred to as GA-treated.
Determination of cell viability
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, Thornwood, NJ, USA) and images were captured using Kodak Ektachrome 160T film (Eastman Kodak, Rochester, NY, USA). A template containing five separate regions was superimposed on these images and live and dead cells in each region were counted. Viability of protoplasts was determined by counting the number of live and dead cells in a sample. Protoplasts were scored dead when the PM became ruffled and, in some experiments, the nucleus stained blue with 4',6-diamino-2-phenylindole dihydrochloride (DAPI, Molecular Probes).
H2O2-induced death of protoplasts
Washed protoplasts (5 µl) were added to 2 ml of baseline culture medium containing 3.25 mM or 325 mM H2O2. The sample of protoplasts was scanned at 20 min (325 mM H2O2) or 60 min (3.25 mM 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.
Ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) activity gels
Aleurone layers (10) were ground to a fine powder in liquid N2, extracted in 300 µl buffer (60 mM K2HPO4, pH 7.8, 0.1 mM EDTA, 20 µM E64, 20 µM pepstatin, 20 µM leupeptin) and the homogenate was centrifuged for 15 min at 4600 rpm at 4 °C. Samples of the homogenate were separated on 12.5% native PAGE at 100 V. SOD activity was assayed using the method as described (Beauchamp and Fridovich, 1971). After electrophoresis gels were immersed in 2.45 mM nitro blue tetrazolium (Sigma, St Louis, MO, USA) for 20 min and soaked in a solution containing 28 mM (TEMED) (tetramethylethylenediamine, TEMED, Sigma), 28 µM riboflavin (Sigma) and 36 mM K2HPO4, pH 7.8 for 15 min. SOD activity was detected by illuminating the gel which causes it to turn uniformly blue except at positions exhibiting SOD activity. When maximum contrast was achieved, the reaction was stopped by rinsing the gel with H2O.
CAT activity was determined after electrophoresis at 100 V and 4 °C for 6 h. The gel was soaked in ddH2O for 15 min. Subsequently, the gel was incubated in 0.03% H2O2 for 10 min. The gel was then carefully washed with ddH2O to remove the residual H2O2. CAT activity was detected by soaking the gel in 1% (w/v; final concentration) ferric chloride and 1% (w/v; final concentration) potassium ferricyanide. This caused the gel to turn uniformly blue except at positions exhibiting CAT activity. When maximum contrast was achieved the reaction was stopped by rinsing the gel with H2O.
APX activity was assayed using the method described previously (Mittler and Zilinskas, 1993). After electrophoresis the gel was immersed in 50 mM sodium phosphate, pH 7.0 and 2 mM ascorbate for 30 min, changing the solution every 10 min. The gel was soaked in 50 mM sodium phosphate, pH 7.0, 4 mM ascorbate and 2 mM H2O2 for an additional 20 min before briefly washing with 50 mM sodium phosphate, pH 7.0. Finally, the gel was incubated in 50 mM sodium phosphate, pH 7.8, 28 mM TEMED (Sigma) and 2.45 mM nitro blue tetrazolium (Sigma) until the gel turned uniformly blue except at positions exhibiting APX activity. When maximum contrast was achieved, the reaction was stopped by rinsing the gel with H2O.
RNA isolation and RNA gel blotting
Aleurone layers (10) were ground to a fine powder in liquid N2 and total barley aleurone RNA was isolated according to the manufacturer's instruction using the Qiagen RNeasy plant mini kit (Qiagen, Inc., Valencia, CA). RNA gel blotting was performed as described in detail elsewhere (Fath et al., 2001). The amount of 32P-labelled cDNA probe hybridized to specific mRNA was determined using a Phosphor Imager (Molecular Dynamics Inc.). All blots were stripped and reprobed with maize 28s rRNA cDNA probe to standardize loading.
Immunoblotting
Aleurone layers were homogenized as described above. Samples (25 µl) of the supernatant were separated by SDS-PAGE and protein blotting was carried out as described in detail (Fath et al., 2001). Secondary antibody (goat-anti-rabbit IgG) coupled to horseradish peroxidase (Sigma, St Louis, MO, USA) was visualized chromogenically.
Light treatments
Protoplasts were illuminated and observed with a Nikon Diaphot microscope (Tokyo, Japan) equipped with either a xenon or mercury bulb (Ushio, Tokyo, Japan) and Nikon 40xHoffman objective. Excitation filters, dichroic mirrors and long pass emission filters were either 370–390 nm excitation, 400 nm dichroic, and 410 nm emission; 330–385 nm excitation, 400 nm dichroic, and 410 nm emission; or 440–495 nm excitation, 500 nm dichroic, and 510 nm emission. All manipulations were performed under dim room light as described earlier (Bethke and Jones, 2001).
An action spectrum for light-induced death was carried out using excitation filters and neutral density filters to vary the excitation wavelength and keep peak illumination intensity constant (Bethke and Jones, 2001). Light was from a xenon bulb and in all cases, intensity was equal to or less than 0.72 W cm-2, with 0.07 W cm-2 at 380 nm and 0.72 W cm-2 at 440–495 nm.
In vivo H2O2 measurements
Protoplasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di(acetoxymethly ester) (CDCDHFDA-AM, Molecular Probes) as described in (Bethke and Jones, 2001). Loaded cells were illuminated using the mercury light source on a Zeiss Axiophot microscope fitted with a 20xNeofluar objective and optical filters (495 nm excitation, 505 nm dichroic mirror, 535+25 emission; Chroma Technology Corp., Brattleboro, VT, USA). Data were analysed and captured as described previously (Bethke and Jones, 2001).
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Results |
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H2O2 kills GA-treated but not ABA-treated aleurone cells
Incubation of aleurone layers or protoplasts in H2O2 brings about rapid cell death of GA-, but not ABA-treated, cells. The rate of cell death varies as a function of H2O2 concentration. When GA-treated protoplasts were incubated in 3.25 mM H2O2, 50% of cells were dead by 4 h of incubation whereas when H2O2 concentration was raised to 325 mM it took only 30 min to kill 50% of cells. Cells of intact aleurone layers are also killed by added H2O2 and, as with protoplasts, sensitivity of cells in the layer to H2O2 depends on prior exposure to GA. Cells of ABA-treated layers do not die when exposed to 325 mM H2O2 for 1 h although all GA-treated cells die after 1 h in 325 mM H2O2 (Bethke and Jones, 2001
).
ABA enhances the metabolism of H2O2 in aleurone protoplasts
The intracellular H2O2 concentration in barley aleurone protoplasts was monitored using the fluorescent probe CDCDHFDA. This probe does not fluoresce until it reacts with H2O2 and it can be loaded into aleurone protoplasts non-invasively without affecting the viability of the cell or the response to hormones. When aleurone protoplasts loaded with CDCDHFDA were exposed to 3.25 mM H2O2 there was an immediate rise in intracellular fluorescence from the probe (Fig. 1). This experiment emphasizes two important points. First, CDCDHFDA can be used to report intracellular H2O2 in aleurone cells and, second, aleurone cells are freely permeable to H2O2. It was also shown that there was a marked difference in fluorescence when GA-treated and ABA-treated cells were loaded with CDCDHFDA and exposed to 3.25 mM H2O2. The magnitude of the signal from the probe was much greater from GA-treated cells than from cells incubated in ABA, suggesting that metabolism of H2O2 is more efficient in ABA-treated protoplasts (Fig. 1).
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Blue and UV-A light elevate the concentration of intracellular H2O2 and kill GA-treated, but not ABA-treated, aleurone cells
High intensity (0.1–0.7 W cm-2) blue and UV-A light also bring about rapid cell death of barley aleurone protoplasts that have been incubated in GA for 4 d or longer. Protoplasts incubated in ABA for 4 d or longer are refractory to blue and UV-A light and remain alive. The action spectrum for light-induced cell death of GA-treated protoplasts shows maxima between 440 and 490 nm and 370–390 nm, and resembles the absorption spectrum for flavins. Support for a role of flavins as the source of AOS in irradiated aleurone cells comes from experiments with KI, an inhibitor of flavin action (Song and More, 1968; Schmidt et al., 1977). When GA-treated aleurone protoplasts were incubated with 1 mM KI, UV-A induced PCD was delayed (Table 1).
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UV-A light was much more effective in promoting cell death of GA-treated cells than was blue light. At comparable intensities, the exposure time for 50% dead GA-treated cells was much shorter for UV-A than blue light. In one typical experiment, 50% of GA-treated cells were dead within 35 min of exposure to 370–390 nm light, whereas it took 55 min of exposure to blue light to kill 50% of cells.
Cell death brought about by light is indistinguishable from PCD brought about by GA (Fig. 2). Bright field microscopy was used to observe light-induced cell death in real time and it was shown that death of individual cells was rapid and accompanied by a change in the permeability of the plasma membrane (PM) to vital stains such as DAPI or YO-PRO-1. The change in permeability of the PM was followed by a ruffling of the PM indicating that the turgor pressure of the cell had been lost (Fig. 2). The morphological features of light-induced death (Fig, 2B) were identical to those observed when cells died after prolonged incubation in GA (Fig. 2A). Cells incubated in ABA and exposed to high intensity blue or UV-A light did not die and showed none of the features described for GA-treated cells (Bethke and Jones, 2001).
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H2O2-sensitive probes were used to determine whether blue and UV-A light killed aleurone cells by increasing intracellular H2O2 concentrations. When GA- or ABA-treated aleurone protoplasts were loaded with CDCDHFDA and exposed to 460–500 nm blue light, there was an immediate increase in signal from the fluorescent probe indicating a sharp rise in intracellular H2O2 (Fig. 3
). Similar data were obtained when protoplasts were irradiated with UV-A light (data not shown). When ABA-treated protoplasts were loaded with CDCDHFDA and irradiated with blue light the magnitude of the fluorescence signal was much lower than that from protoplasts that had been incubated in GA (Fig. 3). These results indicate that intracellular H2O2 concentrations increase following irradiation and that ABA-treated cells are either better able to metabolize H2O2 or produce less of this AOS than GA-treated cells.
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PCD in aleurone is slowed by antioxidants
Because experiments with fluorescent probes point to a role for H2O2 in both hormonally-regulated and light-induced cell death, experiments were carried out with antioxidants and reducing agents to ask whether these compounds could slow the rate of cell death. To test the effects of these compounds on light-induced death, aleurone protoplasts were first incubated in GA for 5–7 d and preincubated in either 50 mM ascorbate for 15 min, 5 mM DTT for 10 min or 0.5 mM BHT for 10 min prior to exposure to UV-A light. Table 1
shows that these compounds had a dramatic effect on cell death when expressed as time for 50% cell death. GA-treated aleurone cells that were incubated in antioxidants or reductants took almost twice as long as control cells for 50% cell death when exposed to UV-A. A similar experiment was carried out to determine whether the antioxidant BHT would slow the rate of death brought about by prolonged incubation in GA alone. Aleurone protoplasts were incubated in GA for 3 d, and the number of living cells determined. Incubation was continued for two additional days in GA in the presence or absence of 100 µM BHT. After 3 d in GA alone there were 2x105 live cells per flask. Incubation in GA for a further 2 d lowered this number to 5x104 cells, a 75% reduction in the number of live cells. When BHT was added, 1x105 cells survived, twice as many as in the absence of BHT (Bethke and Jones, 2001).
Enzymes of AOS metabolism are hormonally regulated in barley aleurone
These experiments with H2O2 and with blue and UV-A light show that GA sensitizes the aleurone cell to these treatments. ABA-treated aleurone cells, on the other hand, are refractory to added H2O2 and irradiation. These results suggest that ABA-treated aleurone cells more effectively metabolize AOS. This hypothesis was tested by measuring steady-state amounts of RNA encoding various AOS metabolizing enzymes. Figure 4 shows results from a time-course experiment where RNA was isolated from barley aleurone layers incubated in GA or ABA for up to 24 h, separated by electrophoresis, blotted onto nylon and sequentially probed with cDNAs for barley catalase (Cat2), and rice ascorbate peroxidase c2 (Apxc2) and superoxide dismutase (Sodc1). The blot was finally probed with a maize rRNA cDNA to check for loading of RNA. Cell death begins 24 h after incubation of barley aleurone layers in GA (Fath et al., 2001), but GA treatment brings about a decline in the steady-state amount of Cat2, Apxc2 and Sodc1 mRNAs 6–12 h after GA treatment. Cat2 and Sodc1 are expressed at high levels in freshly isolated layers, but the amounts of their mRNAs decline with time in GA. For Cat2 mRNA amounts fall below the level of detection after 12 h in GA and Apxc2 and Sodc1 mRNAs are barely detectable by 18 h of incubation in GA (Fig. 4).
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The effect of ABA on Cat, Apx and Sod mRNA is dramatically different from that of GA (Fig. 4
). ABA treatment of aleurone layers results in an increase in the steady-state amount of Cat2, Sodc1 and Apxc2 mRNA. In aleurone layers treated with ABA for 24 h Cat2 and Sod1 mRNAs are more abundant than in GA-treated layers at any time (Fig. 4).
Protein blots (Fig. 5) and enzyme assays (Fig. 6) were used to quantify the amounts and activities of APX, CAT and SOD. Data from these experiments are complementary to those from the RNA blotting experiments and show that neither the amounts of APX, CAT or SOD proteins nor their activities decline when aleurone layers are incubated in ABA for up to 48 h (Fig. 5). The amounts and activities of APX, CAT and SOD all decline following treatment of aleurone layers with GA. These proteins were not detected in aleurone layers incubated in GA for 36 h (Figs 5, 6) even though approximately 50% of the cells in the layer are alive at that time (Fath et al., 2001).
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Aleurone cells have abundant mitochondria, glyoxysomes and stored lipid reserves
Because these experiments with hormones and light implicate AOS in aleurone PCD, a start has been made to characterize the organelles where AOS are synthesized. Mitochondria are abundant in freshly isolated aleurone cells (Jones, 1969b) and density gradient centrifugation of organelles from aleurone layers shows that the number of mitochondria increase during imbibition of the mature, dry, half grains in H2O (Jones, 1980). After incubation in GA for 18 h, as judged by succinate dehydrogenase activity, the number of mitochondria declines by about 50% in layers incubated in GA for 18 h relative to controls incubated in the absence of GA (Jones, 1980). These results have been confirmed and extended in the authors' laboratory by using protein blotting to monitor the abundance of mitochondrial marker proteins. These data show that, whereas marker proteins decline during the first 24 h of incubation in GA, they are not lower in mitochondria of cells incubated in ABA (A Fath and R Jones, unpublished observations).
The seeds of many dicots and monocots show a switch in the electron transport pathway from a CN--insensitive to a CN-sensitive pathway during the early phases of germination and seedling growth (Yentur and Leopold, 1976). Respiratory activity in intact barley aleurone layers was measured and 2 mM KCN was used to block the activity of the cytochrome c oxidase pathway. Aleurone layers incubated in ABA for up to 48 h were relatively insensitive to KCN whereas KCN inhibited O2 consumption of layers treated with GA for 24 h by 35–75% (A Fath and R Jones, unpublished data). Thus, although the number of mitochondria appears to decrease following treatment with GA, the electron transport pathway utilized by these mitochondria switches to one that may increase the rate of AOS production.
Lipid-storing oleosomes are prominent organelles in barley aleurone cells (Jones, 1969a). With increased duration of incubation in GA the number of these organelles declines when compared to the number of oleosomes in cells incubated in H2O (A Fath and R Jones, unpublished data). This reduction in oleosome number correlates with an increase in the activity of glyoxylate cycle enzymes in barley (Jones, 1972; Holtman et al., 1994) and wheat (Doig et al., 1975) and suggests that glyoxysomes are a significant source of AOS in aleurone cells.
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Discussion |
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The data show that AOS play a key role in hormonally regulated PCD in barley aleurone. Aleurone layers are committed to undergo PCD by treatment with GA. Shortly before death intracellular AOS concentrations rise as a result of continuing or enhanced synthesis of AOS coupled with decreasing activities of enzymes that can metabolize AOS. Lowering the activities of AOS metabolizing enzymes in GA-treated cells is manifested by a marked sensitivity of these cells to applied H2O2. By contrast, aleurone cells incubated in ABA do not undergo PCD, and have higher activities of AOS metabolizing enzymes relative to cells that were incubated in GA. The presence of high concentrations of AOS-metabolizing enzymes in ABA-treated cells makes them refractory to high concentration of added H2O2.
Where are AOS synthesized in aleurone cells? The sensitivity of GA-treated aleurone cells to blue and UV-A light point to an important role of flavins in AOS production. First, direct evidence for a role of blue and UV-light in the production of AOS comes from experiments using the H2O2-sensitive dye CDCDHFDA. When aleurone protoplasts were loaded with CDCDHFDA and illuminated there was an immediate and rapid rise in fluorescence, indicating that H2O2 was accumulating in the cell. Second, the action spectrum of light-induced PCD is similar to that for flavins, and the high fluence of the light response suggested photochemical production of H2O2 (Bethke and Jones, 2001). When protoplasts were treated with iodide, a known inhibitor of flavin action (Schmidt et al., 1977) and a quencher of the triplet state of flavins (Song and More, 1968) light-induced death was strongly inhibited.
These experiments on the effects of blue and UV-A light on PCD in aleurone cells have parallels with a similar study on the effects of light on cultured mammalian cells (Hockberger et al., 1999). When 3T3 and CV1 cells were irradiated with blue or violet light there was an increase in AOS production measured by fluorescent probes (Hockberger et al., 1999). Hockberger et al. proposed that light was being absorbed by flavin containing oxidases in the peroxisome and this resulted in a two electron reduction of O2 to H2O2 (Hockberger et al., 1999). GA-treated aleurone cells have abundant glyoxysomes and are likely to contain the flavoprotein fatty acyl CoA oxidase if ß-oxidation of fatty acids occurs (Beevers, 1979). It was hypothesized that this flavoprotein may be the source of AOS when aleurone protoplasts are illuminated with blue or UV-A light.
ß-oxidation and the glyoxylate cycle generate large amounts of NADH that are oxidized in mitochondria (Beevers, 1979). Mitochondria are abundant in the aleurone cells and their numbers increase during imbibition (Jones, 1980). Organelle fractionation experiments have shown that during incubation of isolated aleurone layers in GA the number of mitochondria is reduced (Jones, 1980) and microscopy suggests that during incubation in GA the number of organelles in aleurone cells including mitochondria are dramatically reduced. Freshly isolated aleurone cells are densely packed with membraneous organelles, but during incubation in GA almost all of these organelles are lost.
An important feature of mitochondria in seeds is the extent to which they respire along the alternate oxidase pathway. It is well established that respiration in dormant seeds is resistant to inhibition by CN- and, furthermore, that germination can be promoted by CN- (Taylorson and Hendricks, 1973). Seeds can also switch from the CN--insensitive respiration pathway to one which is inhibited by CN- during germination (Yentur and Leopold, 1976). These results, showing that barley aleurone layers also switch from a respiratory pathway that is CN--insensitive when incubated in ABA to one that becomes sensitive to CN- when incubated in GA, is also interesting in the context of AOS production. Operation of the alternative oxidase pathway in cultured tobacco cells reduces the production of AOS (Maxwell et al., 1999) and it is hypothesized that by maintaining the alternate oxidase pathway in aleurone cells, ABA helps maintain low AOS concentrations.
Taken together, these observation point to metabolic pathways in GA-treated aleurone cells that have the capacity to produce AOS at high rates. This contrasts with the metabolic pathways in ABA-treated aleurone cells where fat metabolism is low, glyoxysomes are much less abundant and mitochondria can utilize an electron transport pathway that minimizes AOS production. It is speculated that one of the roles of ABA in dormant seeds and grains is to repress those metabolic pathways that generate AOS and hence enhance the seed's ability to survive and germinate (Bewley, 1997).
In addition to repressing metabolic pathways that produce AOS, ABA up-regulates the synthesis of enzymes that metabolize AOS. It has been shown that mRNAs encoding CAT, APX and SOD as well as the activities of these enzymes are up-regulated by ABA. CAT activity has been measured in aleurone layers incubated in ABA for up to 5 d and these data confirm what is reported in Figs 4–6. CAT activity increased more than 16-fold after incubation in ABA for 5 d, but after only 1 d in GA, CAT activity was near the lower limit of detection (Fath et al., 2001).
The mechanism whereby ABA and GA regulate steady-state amounts of Apxc2, Cat2 and Sodc1 mRNAs in barley is not known. The promoter of maize Cat2 has a putative GA response element (Guan and Scandalios, 1996) whereas maize Cat1 has a functional ABA response element and all maize catalase genes have putative antioxidant response elements (Guan et al., 2000). Although the data from maize indicate that barley Cat genes may have response elements that allow for fine tuning of their expression by ABA and GA, it is possible that the amounts of Cat mRNAs are regulated post-transcriptionally. Less is known about how genes encoding APX and SOD are regulated in cereals, although there is evidence for hormonal and AOS regulation. ABA has been shown to regulate the expression of Sod4 in maize (Zhu and Scandalios, 1994) and Sodc2 genes in rice (Sakamoto et al., 1995) and cytosolic Apx transcripts were shown to be increased in suspension-cultured rice embryo cells treated with H2O2 (Morita et al., 1999).
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This work was funded by grants to Russell Jones from the National Science Foundation (grant No. IBN-9818047) and the Torrey Mesa Research Institute.
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1 To whom correspondence should be addressed. Fax: +1 510 642 4995. E-mail: rjones{at}nature.berkeley.edu
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