Programmed Cell Death In Plant Cells Research Articles

Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita.

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

Papain-like cysteine proteases are involved in programmed cell death in plants under heavy metal stress

Programmed cell death (PCD) plays a crucial role in plant development (dPCD) and is also activated under the influence of environmental factors (ePCD). As the mechanism of PCD under heavy metal stress is poorly understood, a suspension culture of cells from the facultative metallophyte Viola tricolor was used as a model to study the role of caspase-like proteases in PCD in plant cells under heavy metal stress.Cell suspension cultures treated with Zn or Pb (2000 μM for 72 h) were subjected to TUNEL assay and ligand blotting using a biotinylated inhibitor of caspase-like proteases (biotin-xVAD-fmk). The results from the TUNEL assay showed that the frequency of PCD events was significantly higher in the metal-treated suspended cells (6.8–19.6 % vs 21.8–30.1 %, Zn or Pb, respectively) than it was in the control cells (7.1–9.1 %). The results from the biotinylated inhibitor blot analysis revealed that papain-like cysteine proteases (PLCPs) were involved in heavy metal-induced PCD. Exogenous application of the PLCPs inhibitor E-64-d at a concentration of 50 μM together with Zn or Pb remarkably increased cell viability.This is the first report on the role of the PLCPs in ePCD in plant cells under heavy metal stress.

Cylindrospermopsin induces biochemical changes leading to programmed cell death in plants.

In the present study we provide cytological and biochemical evidence that the cyanotoxin cylindrospermopsin (CYN) induces programmed cell death (PCD) symptoms in two model vascular plants: the dicot white mustard (Sinapis alba) and the monocot common reed (Phragmites australis). Cytological data include chromatin fragmentation and the increase of the ratio of TUNEL-positive cells in roots, the latter being detected in both model systems studied. The strongest biochemical evidence is the elevation of the activity of several single-stranded DNA preferring nucleases-among them enzymes active at both acidic and alkaline conditions and are probably directly related to DNA breaks occurring at the initial stages of plant PCD: 80kDa nucleases and a 26kDa nuclease, both having dual (single- and double-stranded nucleic acid) specificity. Moreover, the total protease activity and in particular, a 53-56kDa alkaline protease activity increases. This protease could be inhibited by PMSF, thus regarded as serine protease. Serine proteases are detected in all organs of Brassicaceae (Arabidopsis) having importance in differentiation of specialized plant tissue through PCD, in protein degradation/processing during early germination and defense mechanisms induced by a variety of biotic and abiotic stresses. However, knowledge of the physiological roles of these proteases and nucleases in PCD still needs further research. It is concluded that CYN treatment induces chromatin fragmentation and PCD in plant cells by activating specific nucleases and proteases. CYN is proposed to be a suitable molecule to study the mechanism of plant apoptosis.

Oxidative stress and mitochondrial dysfunctions are early events in narciclasine‐induced programmed cell death in tobacco Bright Yellow‐2 cells

Narciclasine (NCS) is a plant growth inhibitor isolated from the secreted mucilage of Narcissus tazetta bulbs. It is a commonly used anticancer agent in animal systems. In this study, we provide evidence to show that NCS also acts as an agent in inducing programmed cell death (PCD) in tobacco Bright Yellow-2 (TBY-2) cell cultures. NCS treatment induces typical PCD-associated morphological and biochemical changes, namely cell shrinkage, chromatin condensation and nuclear DNA degradation. To investigate possible signaling events, we analyzed the production of reactive oxygen species (ROS) and the function of mitochondria during PCD induced by NCS. A biphasic behavior burst of hydrogen peroxide (H(2)O(2)) was detected in TBY-2 cells treated with NCS, and mitochondrial transmembrane potential (MTP) loss occurred after a slight increase. Pre-incubation with antioxidant catalase (CAT) and N-acetyl-L-cysteine (NAC) not only significantly decreased the H(2)O(2) production but also effectively retarded the decrease of MTP and reduced the percentage of cells undergoing PCD after NCS treatment. In conclusion, our results suggest that NCS induces PCD in plant cells; the oxidative stress (accumulation of H(2)O(2)) and the MTP loss play important roles during NCS-induced PCD.

Evidence of ceased programmed cell death in metaphloem sieve elements in the developing caryopsis of Triticum aestivum L.

Transmission electron microscopy (TEM) and fluorescence microscopy studies revealed that the metaphloem sieve elements (MSEs) in the ventral vascular bundle of the caryopses of developing wheat (Triticum aestivum L.) undergo a unique type of programmed cell death (PCD). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei were observed at 3 and 4 days after flowering (DAF). Transmission electron microscopy studies of differentiating MSEs revealed increased vacuolation, nuclear degeneration, chromatin condensation and localization to the periphery of the nucleus, and partly dilated perinuclear spaces, all typical characteristics of PCD in plant cells. In addition, vacuoles were disrupted at the last stages of differentiation. These results demonstrate that MSE differentiation is a unique type of PCD with highly selective autophagic processes, in which PCD ceases just prior to death. During this cessation of PCD, vacuoles and the endoplasmic reticulum appear to be associated with selective organelle digestion.

Mitochondria-Associated Hexokinases Play a Role in the Control of Programmed Cell Death inNicotiana benthamiana

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.

Thaxtomin A induces programmed cell death in Arabidopsis thaliana suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.

Activation of the Programmed Cell Death Pathway by Inhibition of Proteasome Function in Plants

Proteasomes constitute the major machinery to degrade or process proteins by ATP/ubiquitin-mediated proteolysis. Recent findings suggest a pivotal role of the ubiquitin/proteasome pathway in the regulation of apoptosis in animal cells. Here we show that virus-induced gene silencing of two different subunits of the 26 S proteasome, the alpha 6 subunit of the 20 S proteasome and RPN9 subunit of 19 S regulatory complex, both activated the programmed cell death (PCD) program, accompanied by reduced proteasome activity and accumulation of polyubiquitinated proteins. These results demonstrate that disruption of proteasome function leads to PCD in plant cells. The affected cells showed morphological markers of PCD, including nuclear condensation and DNA fragmentation, accompanied by the 10-fold higher production of reactive oxygen species and increased ion leakage for 3-fold. Similar to apoptosis in animal system, mitochondrial membrane potential was decreased, cytochrome c released from mitochondria to cytosol, and caspase 9- and caspase 3-like proteolytic activities detected in the cells. Interestingly, this proteasome-mediated PCD stimulated the expression of only a subset of transcripts that are highly induced during pathogen-mediated hypersensitive response cell death, indicating that the two PCD pathways are differentially regulated. Taken together, these results provide the first direct evidence that proteasomes play a role in the regulatory program of PCD in plants. Controlled inhibition of proteasome activities may be involved in developmentally or environmentally activated plant cell death programs.

Constitutive caspase-like machinery executes programmed cell death in plant cells

The morphological features of programmed cell death (PCD) and the molecular machinery involved in the death program in animal cells have been intensively studied. In plants, cell death has been widely observed in predictable patterns throughout differentiation processes and in defense responses. Several lines of evidence argue that plant PCD shares some characteristic features with animal PCD. However, the molecular components of the plant PCD machinery remain obscure. We have shown that plant cells undergo PCD by constitutively expressed molecular machinery upon induction with the fungal elicitor EIX or by staurosporine in the presence of cycloheximide. The permeable peptide caspase inhibitors, zVAD-fmk and zBocD-fmk, blocked PCD induced by EIX or staurosporine. Using labeled VAD-fmk, active caspase-like proteases were detected within intact cells and in cell extracts of the PCD-induced cells. These findings suggest that caspase-like proteases are responsible for the execution of PCD in plant cells.

Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress

Programmed cell death (PCD) may be triggered by a variety of environmental stimuli. In this report we show that low temperature treatment of tobacco BY-2 cells results in specific chromatin changes. The early stage was characterised by chromatin condensation associated with specific endonucleolytic cleavage of the genome into fragments of 50–100 kbp in size. Later, after 2 weeks of the cold treatment, a ladder of nucleosomal units (178 bp) and their multiples occurred. Chromatin changes were accompanied by a general decrease in cell viability. However, the cell culture retained about 11% of living cells even after prolonged incubation in the cold suggesting the presence of a cold-resistant population of cells. The results support the view that PCD was activated by the cold stress. We suggest that cold-stressed tobacco BY-2 culture might be a useful system for investigation of PCD in plant cells.