Genetic buffering of cyclic AMP in Arabidopsis thaliana compromises the plant immune response triggered by an avirulent strain of Pseudomonas syringae pv. tomato.

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non-specific pharmacological approaches. We therefore developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the 'cAMP-sponge' (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with wild-type (WT) plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24 h post-infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium-calmodulin-binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis-regulation of AtSR1 activity, affected the speed and strength of the immune response.

ROS and redox balance as multifaceted players of cross-tolerance: epigenetic and retrograde control of gene expression.

Retrograde pathways occurring between chloroplasts, mitochondria, and the nucleus involve oxidative and antioxidative signals that, working in a synergistic or antagonistic mode, control the expression of specific patterns of genes following stress perception. Increasing evidence also underlines the relevance of mitochondrion-chloroplast-nucleus crosstalk in modulating the whole cellular redox metabolism by a controlled and integrated flux of information. Plants can maintain the acquired tolerance by a stress memory, also operating at the transgenerational level, via epigenetic and miRNA-based mechanisms controlling gene expression. Data discussed in this review strengthen the idea that ROS, redox signals, and shifts in cellular redox balance permeate the signalling network leading to cross-tolerance. The identification of specific ROS/antioxidative signatures leading a plant to different fates under stress is pivotal for identifying strategies to monitor and increase plant fitness in a changing environment. This review provides an update of the plant redox signalling network implicated in stress responses, in particular in cross-tolerance acquisition. The interplay between reactive oxygen species (ROS), ROS-derived signals, and antioxidative pathways is also discussed in terms of plant acclimation to stress in the short and long term.

Low concentrations of the toxin ophiobolin A lead to an arrest of the cell cycle and alter the intracellular partitioning of glutathione between the nuclei and cytoplasm.

Ophiobolin A, a tetracyclic sesterpenoid produced by phytopathogenic fungi, is responsible for catastrophic losses in crop yield but its mechanism of action is not understood. The effects of ophiobolin A were therefore investigated on the growth and redox metabolism of Tobacco Bright Yellow-2 (TBY-2) cell cultures by applying concentrations of the toxin that did not promote cell death. At concentrations between 2 and 5 µM, ophiobolin A inhibited growth and proliferation of the TBY-2 cells, which remained viable. Microscopic and cytofluorimetric analyses showed that ophiobolin A treatment caused a rapid decrease in mitotic index, with a lower percentage of the cells at G1 and increased numbers of cells at the S/G2 phases. Cell size was not changed following treatment suggesting that the arrest of cell cycle progression was not the result of a block on cell growth. The characteristic glutathione redox state and the localization of glutathione in the nucleus during cell proliferation were not changed by ophiobolin A. However, subsequent decreases in glutathione and the re-distribution of glutathione between the cytoplasm and nuclei after mitosis occurring in control cells, as well as the profile of glutathionylated proteins, were changed in the presence of the toxin. The profile of poly ADP-ribosylated proteins were also modified by ophiobolin A. Taken together, these data provide evidence of the mechanism of ophiobolin A action as a cell cycle inhibitor and further demonstrate the link between nuclear glutathione and the cell cycle regulation, suggesting that glutathione-dependent redox controls in the nuclei prior to cell division are of pivotal importance.

Role of redox homeostasis in thermo-tolerance under a climate change scenario.

BACKGROUND: Climate change predictions indicate a progressive increase in average temperatures and an increase in the frequency of heatwaves, which will have a negative impact on crop productivity. Over the last decade, a number of studies have addressed the question of how model plants or specific crops modify their metabolism when exposed to heat stress. SCOPE: This review provides an overview of the redox pathways that contribute to how plants cope with heat stress. The focus is on the role of reactive oxygen species (ROS), redox metabolites and enzymes in the signalling pathways leading to the activation of defence responses. Additional attention is paid to the regulating mechanisms that lead to an increase in specific ROS-scavenging systems during heat stress, which have been studied in different model systems. Finally, increasing thermo-tolerance in model and crop plants by exposing them to heat acclimation or to exogenous treatments is discussed. CONCLUSIONS: Although there is clear evidence that several strategies are specifically activated according to the intensity and the duration of heat stress, as well as the capacity of the different species or genotypes to overcome stress, an alteration in redox homeostasis seems to be a common event. Different mechanisms that act to enhance redox systems enable crops to overcome heat stress more effectively. Knowledge of thermo-tolerance within agronomic biodiversity is thus of key importance to enable researchers to identify new strategies for overcoming the impacts of climate change, and for decision-makers in planning for an uncertain future with new choices and options open to them.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

BACKGROUND AND AIMS: Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. METHODS: Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. KEY RESULTS: Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. CONCLUSIONS: A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.

S-nitrosylation of ascorbate peroxidase is part of programmed cell death signaling in tobacco Bright Yellow-2 cells.

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H2O2) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H2O2-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H2O2-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H2O2-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H2O2 levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.

Exploring Natural Variations in Arabidopsis thaliana: Plant Adaptability to Salt Stress.

The increase in soil salinization represents a current challenge for plant productivity, as most plants, including crops, are mainly salt-sensitive species. The identification of molecular traits underpinning salt tolerance represents a primary goal for breeding programs. In this scenario, the study of intraspecific variability represents a valid tool for investigating natural genetic resources evolved by plants in different environmental conditions. As a model system, Arabidopsis thaliana, including over 750 natural accessions, represents a species extensively studied at phenotypic, metabolic, and genomic levels under different environmental conditions. Two haplogroups showing opposite root architecture (shallow or deep roots) in response to auxin flux perturbation were identified and associated with EXO70A3 locus variations. Here, we studied the influence of these genetic backgrounds on plant salt tolerance. Eight accessions belonging to the two haplogroups were tested for salt sensitivity by exposing them to moderate (75 mM NaCl) or severe (150 mM NaCl) salt stress. Salt-tolerant accessions were found in both haplogroups, and all of them showed efficient ROS-scavenging ability. Even if an exclusive relation between salt tolerance and haplogroup membership was not observed, the modulation of root system architecture might also contribute to salt tolerance.

Fructan metabolism in developing wheat (Triticum aestivum L.) kernels.

Although fructans play a crucial role in wheat kernel development, their metabolism during kernel maturation is far from being understood. In this study, all major fructan-metabolizing enzymes together with fructan content, fructan degree of polymerization and the presence of fructan oligosaccharides were examined in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity. Fructan accumulation occurred mainly in the first 2 weeks after anthesis, and a maximal fructan concentration of 2.5 ± 0.3 mg fructan per kernel was reached at 16 days after anthesis (DAA). Fructan synthesis was catalyzed by 1-SST (sucrose:sucrose 1-fructosyltransferase) and 6-SFT (sucrose:fructan 6-fructosyltransferase), and to a lesser extent by 1-FFT (fructan:fructan 1-fructosyltransferase). Despite the presence of 6G-kestotriose in wheat kernel extracts, the measured 6G-FFT (fructan:fructan 6G-fructosyltransferase) activity levels were low. During kernel filling, which lasted from 2 to 6 weeks after anthesis, kernel fructan content decreased from 2.5 ± 0.3 to 1.31 ± 0.12 mg fructan per kernel (42 DAA) and the average fructan degree of polymerization decreased from 7.3 ± 0.4 (14 DAA) to 4.4 ± 0.1 (42 DAA). FEH (fructan exohydrolase) reached maximal activity between 20 and 28 DAA. No fructan-metabolizing enzyme activities were registered during the final phase of kernel maturation, and fructan content and structure remained unchanged. This study provides insight into the complex metabolism of fructans during wheat kernel development and relates fructan turnover to the general phases of kernel development.

The soluble proteome of tobacco Bright Yellow-2 cells undergoing H2O2-induced programmed cell death.

Plant programmed cell death (PCD) is a genetically controlled process that plays an important role in development and stress responses. Reactive oxygen species (ROS) are key inducers of PCD. The addition of 50 mM H2O2 to tobacco Bright Yellow-2 (TBY-2) cell cultures induces PCD. A comparative proteomic analysis of TBY-2 cells treated with 50 mM H2O2 for 30 min and 3 h was performed. The results showed early down-regulation of several elements in the cellular redox hub and inhibition of the protein repair-degradation system. The expression patterns of proteins involved in the homeostatic response, in particular those associated with metabolism, were consistently altered. The changes in abundance of several cytoskeleton proteins confirmed the active role of the cytoskeleton in PCD signalling. Cells undergoing H2O2-induced PCD fail to cope with oxidative stress. The antioxidant defence system and the anti-PCD signalling cascades are inhibited. This promotes a genetically programmed cell suicide pathway. Fifteen differentially expressed proteins showed an expression pattern similar to that previously observed in TBY-2 cells undergoing heat shock-induced PCD. The possibility that these proteins are part of a core complex required for PCD induction is discussed.

A Multifactorial Regulation of Glutathione Metabolism behind Salt Tolerance in Rice.

Knowledge of the stress-induced metabolic alterations in tolerant and sensitive plants is pivotal for identifying interesting traits that improve plant resilience toward unfavorable environmental conditions. This represents a hot topic area of plant science, particularly for crops, due to its implication in food security. Two rice varieties showing dissimilar resistance to salt, Baldo and Vialone Nano, have been studied to investigate the mechanisms underpinning tolerance toward salinity, and these studies have focused on the root system. A detailed analysis of the salt stress-dependent modulation of the redox network is here presented. The different phenotype observed after salt exposure in the two rice varieties is coherent with a differential regulation of cell-cycle progression and cell-death patterns observed at root level. Baldo, the tolerant variety, already showed a highly responsive antioxidative capacity in control conditions. Consistently, stressed Baldo plants showed a different pattern of H2O2 accumulation compared to Vialone Nano. Moreover, glutathione metabolism was finely modulated at transcriptional, post-transcriptional, and post-translational levels in Baldo. These results contribute to highlight the role of ROS and antioxidative pathways as a part of a complex redox network activated in rice toward salt stress.

Comparison between In Vitro Chemical and Ex Vivo Biological Assays to Evaluate Antioxidant Capacity of Botanical Extracts.

The anti-oxidative activity of plant-derived extracts is well-known and confers health-promoting effects on functional foods and food supplements. Aim of this work is to evaluate the capability of two different assays to predict the real biological antioxidant efficiency. At this purpose, extracts from five different plant-derived matrices and commercial purified phytochemicals were analyzed for their anti-oxidative properties by using well-standardized in vitro chemical method (TEAC) and an ex vivo biological assay. The biological assay, a cellular membrane system obtained from erythrocytes of healthy volunteers, is based on the capability of phytochemicals treatment to prevent membrane lipid peroxidation under oxidative stress by UV-B radiation. Plant extracts naturally rich in phenols with different structure and purified phytochemicals showed different in vitro and ex vivo antioxidant capacities. A high correlation between phenolic contents of the plant-derived extracts and their ability to prevent oxidative injuries in a biological system was found, thus underlying the relevance of this class of metabolites in preventing oxidative stress. On the other hand, a low correlation between the antioxidant capacities was shown between in vitro and ex vivo antioxidant assay. Moreover, data presented in this work show how food complex matrices are more effective in preventing oxidative damages at biological level than pure phytochemicals, even if for these latter, the antioxidant activity was generally higher than that observed for food complex matrices.

Different involvement of the mitochondrial, plastidial and cytosolic ascorbate-glutathione redox enzymes in heat shock responses.

Plant survival under heat stress requires the activation of proper defence mechanisms to avoid the impairment of metabolic functions. Heat stress leads to the overproduction of reactive oxygen species (ROS) in the cell. In plants, the ascorbate (ASC)-GSH cycle plays a pivotal role in controlling ROS levels and cellular redox homeostasis. Ascorbate peroxidase (APX) is the enzyme of this cycle mainly involved in ROS detoxification. In this study, the ASC-GSH cycle enzymes were analysed in the cytosol, mitochondria and plastids of tobacco Bright Yellow-2 cultured cells. The cells were also subjected to two different heat shocks (HSs; 35 or 55 degrees C for 10 min) and the cell compartments were isolated in both conditions. The results reported here indicate that moderate HS (35 degrees C) does not affect cell viability, whereas cell exposure to 55 degrees C HS induces programmed cell death (PCD). In relation to ASC-GSH cycle, the three analysed compartments have specific enzymatic profiles that are diversely altered by the HS treatments. The cytosol contains the highest activity of all ASC-GSH cycle enzymes and the data reported here suggest that it acts as a redox buffer for the whole cells. In particular, the cytosolic APX seems to be the most versatile enzyme, being its activity enhanced after moderate HS and reduced during PCD induction, whereas the other APX isoenzymes are only affected in the cells undergoing PCD. The relevance of the changes in the different ASC-GSH cycle isoenzymes in allowing cell survival or promoting PCD is discussed.

Analysis of Redox Relationships in the Plant Cell Cycle: Determination of Ascorbate, Glutathione, and Poly(ADPribose)polymerase (PARP) in Plant Cell Cultures.

Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signalling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced ROS accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly(ADP-ribose)polymerase (PARP) during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

Redox Balance-DDR-miRNA Triangle: Relevance in Genome Stability and Stress Responses in Plants.

Plants are continuously faced with complex environmental conditions which can affect the oxidative metabolism and photosynthetic efficiency, thus leading to the over-production of reactive oxygen species (ROS). Over a certain threshold, ROS can damage DNA. DNA damage, unless repaired, can affect genome stability, thus interfering with cell survival and severely reducing crop productivity. A complex network of pathways involved in DNA damage response (DDR) needs to be activated in order to maintain genome integrity. The expression of specific genes belonging to these pathways can be used as indicators of oxidative DNA damage and effective DNA repair in plants subjected to stress conditions. Managing ROS levels by modulating their production and scavenging systems shifts the role of these compounds from toxic molecules to key messengers involved in plant tolerance acquisition. Oxidative and anti-oxidative signals normally move among the different cell compartments, including the nucleus, cytosol, and organelles. Nuclei are dynamically equipped with different redox systems, such as glutathione (GSH), thiol reductases, and redox regulated transcription factors (TFs). The nuclear redox network participates in the regulation of the DNA metabolism, in terms of transcriptional events, replication, and repair mechanisms. This mainly occurs through redox-dependent regulatory mechanisms comprising redox buffering and post-translational modifications, such as the thiol-disulphide switch, glutathionylation, and S-nitrosylation. The regulatory role of microRNAs (miRNAs) is also emerging for the maintenance of genome stability and the modulation of antioxidative machinery under adverse environmental conditions. In fact, redox systems and DDR pathways can be controlled at a post-transcriptional level by miRNAs. This review reports on the interconnections between the DDR pathways and redox balancing systems. It presents a new dynamic picture by taking into account the shared regulatory mechanism mediated by miRNAs in plant defense responses to stress.

Salt tolerance in indica rice cell cultures depends on a fine tuning of ROS signalling and homeostasis.

Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant's response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant's response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO's possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.

Effects of ionizing radiation on bio-active plant extracts useful for preventing oxidative damages.

Humans are exposed to ionizing radiations in medical radiodiagnosis and radiotherapy that cause oxidative damages and degenerative diseases. Airplane pilots, and even more astronauts, are exposed to a variety of potentially harmful factors, including cosmic radiations. Among the phytochemicals, phenols are particularly efficient in countering the oxidative stress. In the present study, different extracts obtained from plant food, plant by-products and dietary supplements, have been compared for their antioxidant properties before and after irradiation of 140 cGy, a dose absorbed during a hypothetical stay of three years in the space. All the dry extracts, characterized in terms of vitamin C and phenolic content, remained chemically unaltered and maintained their antioxidant capability after irradiation. Our results suggest the potential use of these extracts as nutraceuticals to protect humans from oxidative damages, even when these extracts must be stored in an environment exposed to cosmic radiations as in a space station.

Production of reactive species and modulation of antioxidant network in response to heat shock: a critical balance for cell fate.

Exposure to adverse temperature conditions is a common stress factor for plants. In order to cope with heat stress, plants activate several defence mechanisms responsible for the control of reactive oxygen species (ROS) and redox homeostasis. Specific heat shocks (HSs) are also able to activate programmed cell death (PCD). In this paper, the alteration of several oxidative markers and ROS scavenging enzymes were studied after subjecting cells to two different HSs. Our results suggest that, under moderate HS, the redox homeostasis is mainly guaranteed by an increase in glutathione (GSH) content and in the ascorbate peroxidase (APX) and catalase (CAT) activities. These two enzymes undergo different regulatory mechanisms. On the other hand, the HS-induced PCD determines an increase in the activity of the enzymes recycling the ascorbate- and GSH-oxidized forms and a reduction of APX; whereas, CAT decreases only after a transient rise of its activity, which occurs in spite of the decrease of its gene expression. These results suggest that the enzyme-dependent ROS scavenging is enhanced under moderate HS and suppressed under HS-induced PCD. Moreover, the APX suppression occurring very early during PCD, could represent a hallmark of cells that have activated a suicide programme.

Analysis of redox relationships in the plant cell cycle: determinations of ascorbate, glutathione and poly (ADPribose)polymerase (PARP) in plant cell cultures.

Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signaling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced reactive oxygen species accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly (ADP-ribose) polymerase during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

Programmed Cell Death in Plants: An Overview.

Programmed cell death (PCD) is a controlled mechanism that eliminates specific cells under developmental or environmental stimuli. All organisms-from bacteria to multicellular eukaryotes-have the ability to induce PCD in selected cells. Although this process was first identified in plants, the interest in deciphering the signaling pathways leading to PCD strongly increased when evidence came to light that PCD may be involved in several human diseases. In plants, PCD activation ensures the correct occurrence of growth and developmental processes, among which embryogenesis and differentiation of tracheary elements. PCD is also part of the defense responses activated by plants against environmental stresses, both abiotic and biotic.This chapter gives an overview of the roles of PCD in plants as well as the problems arising in classifying different kinds of PCD according to defined biochemical and cellular markers, and in comparison with the various types of PCD occurring in mammal cells. The importance of understanding PCD signaling pathways, with their elicitors and effectors, in order to improve plant productivity and resistance to environmental stresses is also taken into consideration.

Plant Cell Cultures as Model Systems to Study Programmed Cell Death.

The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.