[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.
[show abstract][hide abstract] ABSTRACT: The aleurone layer of cereals is a secretory tissue whose activity is regulated by abscisic acid (ABA) and gibberellins (GAs).
Whereas GA triggers enzyme synthesis and secretion and initiates a program that culminates in cell death, ABA prevents enzyme
production and cell death. Reactive oxygen species (ROS) are key players in regulating cell viability and GA sensitizes the
aleurone cell to ROS. Sensitivity of GA-treated cells results in part from a reduction in steady-state amounts of mRNAs encoding
enzymes that scavenge ROS. mRNAs encoding catalase, superoxide dismutase and ascorbate peroxidase are almost undetectable
in aleurone layers 24 h after incubation in GA. For layers incubated in ABA, however, the amounts of these mRNAs increase.
Western blotting and enzyme activity assays confirm that GA but not ABA reduced the amount and activity of ROS scavenging
enzymes (Fath et al., 2001b). Substantial amounts of ROS are produced by enzymes engaged in lipid metabolism, and by the electron
transport chain in the mitochondria. Aleurone layers contain abundant stores of triglycerides and ROS are produced as these
lipids are rapidly converted to sugars. We hypothesize that the ROS produced in GA-treated aleurone cells bring about cell
death by disrupting the plasma membrane. Aleurone cells incubated in ABA, on the other hand, are better able to maintain redox
balance. ABA does not initiate rapid triglyceride metabolism, and the activities of ROS-scavenging enzymes remain high in
ABA-treated cells. We conclude that GA initiates a metabolic cascade in aleurone cells that results in death from ROS. ABA
maintains viability by keeping ROS under control.
[show abstract][hide abstract] ABSTRACT: 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 H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) 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 H(2)O(2) 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.
Journal of Experimental Botany 06/2002; 53(372):1273-82. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.AbbreviationsRO, reactive oxygen species; HR, hypersensitive response; PSV, protein storage vacuole; PCD, programmed cell death; CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase.
New Phytologist 06/2001; 151(1):99 - 107. · 6.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, and Cat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.
[show abstract][hide abstract] ABSTRACT: Aleurone cells are essential for the mobilization of reserves stored in the endosperm of cereals but are not required after seedling establishment. Following imbibition the embryo produces gibberellins (GAs) that initiate a program in the aleurone cell that results in the synthesis and secretion of a broad spectrum of acid hydrolases. When the aleurone layer has fulfilled its role as a secretory gland and the contents of its cells are depleted, the aleurone cell dies. Because aleurone cell death is part of the normal development of the grain and is initiated by endogenous hormones, we classify it as a form of programmed cell death (PCD). Abscisic acid (ABA) is produced in dormant and quiescent grain and this hormone prevents the initiation of the GA-triggered cascade that leads to enzyme production and cell death. Based on these data we argue that ABA is a key factor in maintaining aleurone cell viability and preventing PCD in dormant and quiescent grain. Aleurone cells die when ROS accumulate and cause oxidative damage to cell membranes. Aleurone cells store large amounts of triglycerides and the conversion of these lipids to sugars by β-oxidation produces H2O2 and contributes to oxidative stress in the aleurone cell. ROS are normally metabolized by enzymes, including ascorbate peroxidase, catalase and superoxide dismutase. We hypothesize that a reduction in the activities of ROS-metabolizing enzymes and a decrease in the number of mitochondria following GA treatment prevents the aleurone cell from effectively metabolizing ROS. This in turn leads to oxidative damage and cell death.
[show abstract][hide abstract] ABSTRACT: Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.
[show abstract][hide abstract] ABSTRACT: Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.
The Plant Journal 12/1999; 20(3):305-15. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevents PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmarks of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. GA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.
[show abstract][hide abstract] ABSTRACT: Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.
The Plant Cell 06/1999; 11(6):1033-46. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K+ and olfactory channels. Expression of a subclone of amino acids 482-702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K+ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K+ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.
Proceedings of the National Academy of Sciences 03/1998; 95(4):1944-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.
The Plant Cell 01/1997; 8(12):2325-2333. · 9.25 Impact Factor