Programmed cell death in plants - CiteSeerX

9

Journal of Cell and Molecular Biology 4: 9-23, 2005. Haliç University, Printed in Turkey.

P rogrammed cell death in plants Narcin Palavan-Unsal*, Elif-Damla Buyuktuncer and Mehmet Ali Tufekci Halic University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Findikzade 34280, Istanbul-Turkey (* author for correspondence) Abstract Plant development involves the elimination of cell organelles, protoplasts, tissues and organs. Programmed cell death is a process aimed at the removal of redundant, misplaced or damaged cells and maintenance of multicellular organisms. In contrast to the relatively well-described cell death pathway in animals, often referred to as apoptosis, mechanisms and regulation of plant programmed cell death are still well defined. Several morphological and biochemical similarities between apoptosis and plant programmed cell death have been described, including DNA laddering, caspase-like proteolytic activity and cytochrome c release from mitochondria. The aim of this study is to review the examples of programmed cell death through the life cycles of plants and also programmed cell death detection of methods.

Key words: Apoptosis, programmed cell death, plant.

Bitkilerde programlanm›fl hücre ölümü Özet Bitki geliflimi, hücre organellerinin, protoplast, doku ve organlar›n eliminasyonunu içermektedir. Programlanm›fl hücre ölümü gere¤i olmayan, yanl›fl yerleflimi olan ve hasarl› hücrelerin ortadan kald›r›lmas›n› ve çok hücreli organizmalar›n devaml›l›¤›n› sa¤layan bir olayd›r. Hayvanlarda apoptoz olarak bilinen ve çok iyi tan›mlanm›fl hücre ölüm yola¤›n›n aksine bitki programlanm›fl hücre ölümünün mekanizmas› ve düzenlenmesi henüz tam olarak aç›kl›¤a kavuflturulamam›flt›r. Apoptoz ve bitki programlanm›fl hücre ölümü aras›nda DNA fragmentasyonu, kaspazbenzeri proteolitik aktivite ve mitokondrilerden sitokrom c sal›nmas› gibi baz› morfolojik ve biyokimyasal benzerliklerin oldu¤u saptanm›flt›r. Bu çal›flmada bitki yaflam› boyunca meydana gelen programl› hücre ölümlerine örnekler ve ayn› zamanda programlanm›fl hücre ölümünü saptama yöntemlerini derlemek amaç edinildi.

Anahtar sözcükler: Apoptoz, programlanm›fl hücre ölümü, bitki

Introduction The cells of multicellular organisms are members of highly organized community. Controlling the rate of cell division and of cell death strictly regulates the number of cells in this community. If cells are no more needed, they die by activating intracellular death program, for this reason this process named as p rogrammed cell death (PCD) and more commonly

apoptosis. The term apoptosis comes from plant kingdom from old Greek apoptosis that originally means the loss of petals or leaves. Surprisingly, despite the obvious role of cell death in plants the concept of PCD is developed and pioneered in animal and medical sciences. The amount of apoptosis that occurs in developing vertebrate nervous system and adult animal system is astonishing. In the developing vertebrate nervous

10

Narcin Palavan-Unsal et al.

system half or more of the nerve cells normally die soon after the formation. In healthy adult human, every hour billions of cells die in the bone marrow and intestine. What is the purpose of this massive cell death? A molecular mechanism for eliminating developmentally unwanted cells is essential for successful development and growth of complex multicellular organisms. Therefore in addition to regulating the rate of cell division, multicellular organisms such as animals and plants contain a biochemical pathway to control cell death. By coordinating the activation of cell division and cell death, animals and plants may direct a variety of developmental processes such as generation of developmental patterns and the shaping of cells, tissues and organs. However, cell death may not be limited to development and may also be used in a number of other processes such as control of cell populations and defense against invading microbes (Ellis and Horvitz, 1986; Raff, 1992; Greenberg, 1996; Jones and Dangl, 1996; Mittler and Lam, 1996).

Molecular mechanism of apoptosis Cells that die as a result of injury, typically swell and burst and they spill their content all over the

Figure 1: Morphological differences between apoptosis and necrosis (Studzinski, 1999).

neighbors. This process named as cell necrosis, and it causes inflammatory response in animals. By contrast, a cell that undergoes apoptosis dies without damaging neighbors. The cell shrinks and condenses. The cytoskeleton collapses, nuclear envelope dissembles and nuclear DNA breaks up into fragments. Apoptotic bodies that are formed during apoptosis are engulfed

Table 1: Pathological features of apoptosis and necrosis.

Pattern of death Cell size

Plasma membrane

Mitochondria

Organelle Shape Nuclei

DNA Degradation

Cell Degradation

Apoptosis

Necrosis

Single cells Shrinkage Fragmentation Preserved continuity Blebbed Phosphatidylserine on surface Increased membrane permeability Contents released into cytoplasm Cytochrome c; Apaf1 Structure relatively preserved Contracted "Apoptotic bodies" Chromatin: Clumps and Fragmented Fragmented Internucleosomal cleavage Free 3' ends Laddering on electrophoresis DNA appears in cytoplasm Phagocytosis No inflammation

Groups of neighboring cells Swelling Smoothing Early lysis Swelling Disordered structure

Swelling Disruption Membrane disruption

Diffuse and Random

Inflammation Macrophage invasion

Apoptosis in plants

and recycled by neighboring cells or specific macrophages; therefore complete elimination of the cell occurs. The intracellular machinery responsible for apoptosis seems to be similar in all animal cells. This machinery depends on a family of proteases that have a cysteine at their active site, and cleave their target proteins at specific aspartic acids, therefore they are called caspases. Caspases are synthesized in the cell as inactive precursors or procaspases, which are usually activated by cleavage at aspartic acids by other caspases. Once activated, caspases cleave and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade. Some of the activated caspases then cleave other key proteins in the cell. For example some cleave the nuclear lamins and cause irreversible breakdown. Another cleaves protein that normally holds a DNA degrading enzyme in an active form, freeing the DNAase to cut the DNA, thus, cell dismantles itself quickly. First, Uren et al. (2000) have identified genes encoding ancestral caspase-like proteins, the metacaspases, which are present in plants, fungi and protozoa. Homology of metacaspases to caspases is not restricted to the primary sequence, including the catalytic diad of histidine and cysteine, but extends to the secondary structure as well. Recently, Bozhkov et al. (2004) address the question of whether caspase-like proteolytic activity is involved in the regulation of plant developmental cell death using Norway spruce (Picea abies) somatic embryogenesis as the model system. They showed, for the first time, that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity is increased at the early stages of embryo development, and is directly involved in the terminal differentiation and death of the embryo suspensor. Unlike animal cells, plant cells have walls that may act as physical barriers preventing the recycling of cellular material from dead cells via apoptotic bodies. Therefore, recycling of cellular content from dead cells may occur by degradation of cell debris to compounds with low molecular weight and neighbor cells take them. This kind of process releasing cellular debris into the intercellular space would have caused an inflammatory response in animals, but plants are different from animals, because they have no immune response. Morphogenesis in plants is primarily determined by cell division and cell death but no cell

11

migration unlike animal morphogenesis. Another aspect of plant life that involves PCD is the interaction of plants with their environment. Thus the defense of plants against biotic and abiotic stresses often involves activation of apoptosis. Therefore function of cell death is similar but mechanisms concerned are very different and specific for particular organisms.

Molecular markers of apoptosis PCD can be subdivided into three stages: Signaling phase, execution phase and dismantling phase (Depreatere and Golstein, 1998). The regulation of apoptosis is mainly known in neoplastic tissues (Korsmeyer, 1995). Over the past ten years about 30 new molecules have been found that initiate and regulate apoptosis. 20 other molecules associated with signaling or DNA replication, transcription or repair have also been discovered as apoptosis regulators (Willie, 1998). One of the signals for apoptosis is a decrease in mitochondrial transmembrane potential, irrespective of any apoptosis-inducing stimulus (Kroemer et al., 1998). Another early marker of apoptosis is aberrant exposure of phosphatidylserine in the plasma membrane (Kroemer et al., 1998). These events are followed by the activation of proteases, phospholipases and phosphatases. The role of calcium was also well documented (Schwartzman and Cidlowski, 1993). The activation of nucleases leads to cleavage of nuclear DNA (Bayly et al., 1997). Internucleosomal DNA cleavage results in the formation of small fragments (Oberhammer et al., 1993).

Occorunce of apoptosis in plants Plants eliminate cells, organs and parts during responses to stress and expression during various developmental processes:

Apoptosis during reproductive period Unpollinated flowers are fully thrown away. Ovaries with fertilized egg cells in ovules on the same plant are retained forming fruits while the other parts; petals, sepals or tepals fall off. Stigmas and pistils may also be eliminated. In apomictic species, the fruits develop

12


without fertilization, which means that the ovaries with ovules are retained forming fruit, but the other flower parts are eliminated. Apoptosis is involved in the formation of female gametes in seed plants. Single meiotic division gives four haploid megaspore cells, three of them undergo apoptosis, remaining one have two additional mitotic division and bring to egg and associated cells of the embryo (Bell, 1996). Apoptosis is also involved in the formation of male sexual organs. Tapetum layer is surrounding the pollen during maturation undergoes apoptosis (Greenberg, 1996). Plants developed several mechanisms to avoid self-pollination. One of these involves inhibition of germinating pollen dependent on recognition by pistil tissue. This process is mediated by proteins showing RNase activity, which is crucial for their function (Kao and McCubbin, 1996). The growth of the pollen tube through the pistil is associated by selective cell death. Therefore pistil cells along the growth way of the pollen tube undergo apoptosis while the rest of the tissue stays intact (Wang et al., 1996). Two synergid cells are present at the entry to the egg sack, one of them undergoes apoptosis for arriving pollen tube to enter and release sperm cells. Apoptosis also occurs during the embryogenesis in plants. Cell death within the embryo does occur as part of its normal development and includes the death of scutellar cells surrounding the developing radicle, death of suspensor and death of nucellus from which the egg cell originates. These cell types that undergo cell death are highly specific and their death is essential for the final development of the embryo. In addition, in some species the transient endosperm undergoes a cell death that is followed by its reabsorption during embryogenesis that is thought to facilitate embryo growth, whereas in other species in which the endosperm is persistent, it survives as a part of the mature seed. Apoptosis occurs during the germination of plants and it is also formed in the seed storage tissues. Endosperm supplies nutrients to the embryo for development and germination and undergoes PCD. This process generally associated with lytic enzyme activities, for instance α-amylase is secreted from aleurone layer which surrounds the endosperm (Brown and Ho, 1987). Using a model system of barley aleurone protoplasts, Fath et al. (2000) revealed that this PCD occurs in a gibberellic acid dependent manner.

Apoptosis in vegetative plant tissues Generally the structure of most of the leaves is determined by differential cell and tissue growth, but in some genus for instance in Monstera a group of cells die at early stages of leaf development, resulting in the formation of holes in the mature leaf (Kaplan, 1984; Greenberg, 1996). Sclerenchyma cells are dead because thick cell walls perform the mechanical function. Cork is constituted of characteristic cells with thick suberinised layer of the cell wall. Suberin combined with lack of intercellular spaces, protects internal tissues against dessication. The protoplast is no longer needed, therefore it is eliminated. The continuous growth of the stem is also result with the cell death. Cell division in the cambium layer causes cell death in the cork layer, that is replaced with the ruptured epidermis and also in parenchyma cells at the stem pith.

Xylogenesis Perhaps the most dramatic example of PCD is the vascular system differentiation in plants. Tracheal elements (vessels/tracheids) are composed of a series of hollow dead cells. After the formation of secondary walls tracheal elements lose their cellular contents to become empty dead cells. Studies have revealed that this cell death is under spatial and temporal regulation (Fukuda, 1996, 2000). Recent progress in the study of tracheary elements PCD has been made mainly with an in vitro Zinnia system established by Fukuda and Komamine (1980a). In this system single mesophyll cells isolated from Zinnia leaves transdifferentiate synchronously into tracheary elements at a high frequency without cell division (Fukuda and Komamine, 1980a, b). A number of ultrastructural observations of the PCD in tracheal element differentiation have been reported (Obara and Fukuda, 2003). These studies revealed the rapid and progressive cell-autonomous degradation of organelles, including nuclei, vacuoles, plastids, mitochondria and endoplasmic reticulum and at maturity the loss of plasma membrane and some parts of the cell walls. Recently, serial observations of living tracheary elements demonstrated that rapid nuclear degradation is triggered by vacuolar rupture (Obara et al., 2001). Nucleoids in chloroplasts are also degraded rapidly after vacuole rupture. Cytoplasmic streaming ceases immediately after the disruption of

Apoptosis in plants

the vacuole (Groover et al., 1997). All these observations revealed that one of the most critical steps in PCD is the irreversible disruption of tonoplast. Secondary wall lignification is initiated before the vacuole rupture. It was found recently that brassinosteroid biosynthetic pathway is activated before the tracheary element PCD, and the synthesized brassinosteroids induce PCD and the formation of secondary cell walls (Yamamoto et al., 2001). In animals apoptosis usually involves nuclear shrinkage and fragmentation, cellular shrinkage, DNA fragmentation, membrane budding, formation of apoptotic bodies and digestion by macrophages or adjacent cells (Wyllie et al., 1980). But nuclear shrinkage and fragmentation do not occur in tracheary PCD, no prominent chromatin condensation is established, although the nucleus sometimes exhibits chromatin condensation near the nuclear envelope (Lai and Srivastava, 1976; Groover et al., 1997; Obara et al., 2001). Cellular shrinkage, membrane blebbing and the formation of apoptotic bodies do not occur in tracheary element PCD. No DNA ladder has been detected in differentiating tracheal elements. Therefore the morphological features of tracheary elements PCD are different from those of apoptosis. Rapid nuclear degradation after vacuole ruptures implies the involvement of a highly active nuclease. In cultured Zinnia cells at least seven active RNase bands were detected by gel assay (Thelen and Northcote, 1989). Proteases are also involved in the autolytic processes of tracheary element PCD. Several protease activities have been found associated with tracheary element differentiation in the Zinnia system (Obara and Fukuda, 2003). Extracts from Zinnia cells cultured in tracheary element inductive medium contain cystein protease activity (Minami and Fukuda, 1995; Beers and Freeman, 1997). Serine proteases may also be involved in tracheary element PCD. Serine proteases of 145 kDa and 60 kDa have been detected specifically in differentiating tracheary elements (Beers and Freeman, 1997). Many cell death related hydrolytic enzymes are expressed during the autolysis of tracheary elements. These enzymes may be harmful to the other cells if they leak from dead tracheary cells. Therefore, the vascular tissue may have some system by which harmful extracellular enzymes are detoxified.

13

Apoptosis in senescence Senescence in plants can refer to at least two distinct processes: The aging of various tissues and organs as the whole plant matures and the process of the whole plant death that sometimes occurs after fertilization and called as monocarpic senescence (Nooden, 1988). Senescence is a genetically controlled developmental process, which is internally programmed (Nooden and Guiamet, 1996). Ultrastructural researches showed that some features of senescence resemble to the typical markers of PCD. Orzaez and Granell (1997a) established typical DNA fragmentation during the senescence of unpollinated pistils of Pisum sativum. Apoptotic parameters were detected during the petal senescence by Orzaez and Granell (1997b). Same researchers also reported the control of DNA fragmentation by ethylene in connection with senescence. These results provide direct evidence to support that the natural senescence of the leaves is indeed apoptotic process (Yen and Yang, 1998). Several researches have tried to find out molecular approaches to identifying genes involved in senescence control. Several genes termed senescence associated genes (SAG) that show sequence similarity to cysteine proteases induced early senescence (Hensel et al., 1993; Lohman et al., 1994). These plant proteases are good candidates for cell death initiation genes. It has also been suggested that RNase (Blank and McKeon, 1989) and lipoxygenase (Rouet-Mayer et al., 1992) activities are also might be involved in senescence control, since the activity of these enzymes increases during senescence, but no casual link between these activities and senescence has been established, yet. Early researches for genes induced during senescence were unsuccessful to identify transcription factors associated with senescence. But in the last few years with the use of new and powerful techniques, new senescence-associated genes (SAGs) have been identified. A number of potential transcription factors are now known to be associated with senescence (Yang et al., 2001; Zentgraf and Kolb, 2002). Receptor like protein kinases has been concerned in senescence signaling (Robatzek and Somssich, 2002). It is known that receptor like kinases serve as receivers and transducers of external stimuli, acting through phosphorylation/dephosphorylation cascades that lead to changes in gene expression. The

14


Figure 2: A summary of the development of tracheal element PCD. As the PCD process progresses tracheal elements accumulate hydrolytic enzymes in the central vacuole. The transport of organic anions (A-) into the vacuole declines. Tracheal elements become highly vacuolated and their nuclei are tightly pressed and flattened. Secondary cell walls become visible, the central vacuoles in tracheal elements collapse, resulting in the release of hydrolytic enzymes. DNA in tracheal elements is rapidly degraded within 10-20 min of the collapse of the vacuole. After several hours, perforations open at one longitudinal end of each tracheal element, and tracheal elements lose their cellular contents (adapted from Obara and Fukuda, 2003).

senescence associated kinase receptor gene (SARK) behaves as typical SAG that is induced by senescenceinducing factors (ethylene, jasmonate) and repressed by senescence delaying factors (cytokinin, light). Both transcript and protein appear prior to onset of senescence (Hajouj et al., 2000).

P rogrammed cell death in response to abiotic stress Plant cells and tissues exposed to variety of abiotic stresses that ultimately may result in their death. Abiotic stresses include toxins such as salinity, metals, herbicides and gaseous pollutants, including reactive oxygen species (ROS), as well as water deficit and water logging, high and low temperature and extreme illumination. Plants show adaptations to the stress including mechanisms to tolerate the adverse conditions, to exclude the toxins or to avoid conditions where the stress is extreme. Abiotic stress may also result in stunted growth, followed by death of part or

all of the plant. Cell death in abiotic stress may therefore be part of a regulated process to ensure survival. Alternatively, it may be due to the uncontrolled death of cells or tissues killed by unfavorable conditions. PCD may be a part of an adaptive mechanism to survive the stress. Adaptation of plants to environmental conditions such as high light intensity or low humidity often involves covering their surfaces with layer of dead unicellular hairs. These cells are thought to go through PCD resulting in the formation of a protective layer that functions to block high irradiance and trap humidity (Greenberg, 1996). Aerenchyma is the term given to tissues containing gas spaces. It is frequently observed in the roots of wetland species, but may also be formed in some dryland species in unfavorable conditions. It is formed either constitutively or because of abiotic stress, generally originating from water logging. Aerenchyma has been described in two basic types: Lysigenous and

Apoptosis in plants

schizogenous. Lysigenous aerenchyma is formed when previously formed cell die within a tissue to create a gas space. Lysigenous earenchyma is found in rice, wheat, barley and maize (Evans, 2004). Schizogenous aerenchyma is formed when intracellular gas spaces form within a tissue as it develops and without cell death taking place. Spaces are formed by differential growth of adjacent cells with cell separating from each other. Wetland species like Rumex and Sagittaria (Justin and Armstrong, 1987; Schussler and Longstreth, 1996) have characteristic schizegenous aerenchyma that is not involved in the cell death. Recently the plant hormone ethylene was implicated in regulating cell death processes. It is known that hypoxia conditions result in the accumulation of ethylene within the tissue (Jackson et al., 1985). Aerenchyma formation in a member of species can be induced by ethylene produced endogenously (Jackson et al., 1985). This indicates that metabolic consequences of hypoxia are not major factors in cortical cell death and suggests the initiation of a cell death pathway (Gunawardena et al., 2001). Indeed, both an abiotic factor and an endogenous hormone can initiate cell death in these tissues. The first signs of cell death detectable within maize cells treated with ethylene or low oxygen are an invagination of plasma membrane, a more electron dense cytoplasm and shrinkage of plasma membrane from the cell wall (Gunawardena et al., 2001). The granular staining of the vacuolar contents and the

15

formation of numerous vesicles beneath the plasma membrane established. These researchers also revealed wall changes at a very early stage of cell death. Schussler and Longstreth (2000), observed nuclear condensation, which are the characteristics of apoptosis in lysigenous cell death in S. lancifolia. One of the key characteristics of apoptosis is the formation of apoptotic bodies in animal cells. Apoptotic bodies are membrane-bounded inclusions containing chromatin and organelles that remain intact to a late stage in cell death. Membrane bounded inclusions were observed in aerenchyma formation in maize tissues (Gunawardena et al., 2001). The function of these membrane inclusions in plants is not known, they may protect the organelles from lysis or may be involved in maintaining secretion of the enzymes that digest the cell wall and the cytoplasmic contents to form gas spaces. Another characteristic of apoptosis in animal cells is the fragmentation of nuclear DNA. Gunawardena et al. (2001) observed TUNEL-positive (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) nuclei in the cortex of maize roots induced to form aerenchyma by both ethylene and hypoxia. Table 2 summarizes the ultrastructural characterization of PCD in different abiotic stress conditions.

P rogrammed cell death in response to biotic stress Many studies have demonstrated the induction of PCD in plants in response to pathogen attack, indicating that

Table 2: Ultrastructural changes caused by various abiotic stresses (Evans, 2004). Abiotic stress

Ultrastructural changes

Hypoxia-lysigenous

Chromatin condensation and DNA fragmentation

Aerenchyma formation

Organelle surrounded by membranes Plasma membrane invagination and tonoplast degradation Cell wall degradation

Light radiation

Oligonucleosomal fragmentation of DNA Migration of nuclear contents to cell periphery

Mechanical stress

TUNEL positive material around nuclear periphery Oligonucleosomal fragmentation of DNA in chloroplast and nuclei

Cold stress

Chloroplast swelling, thylakoids distort and swell, grana unstuck and chloroplast lyse, nuclei swell, chromatin fragments, ER and golgi cisternae swell, cytoplasmic condensation occurs

16


PCD plays central role in pathogenesis (Goodman and Novacky, 1994). Recent studies showed that cells challenged by pathogens initiate an active PCD response, which is triggered by host-specific signals and requires synthesis of new proteins and/or activation of specific metabolic pathways (He et al., 1994; Greenberg, 1997). At least two types of cell death occur following the infection of a plant with a pathogen: 1. The hypersensitive response (HR). A rapid PCD process that is activated in some plants in order to inhibit the spread of invading pathogen. 2. Disease symptoms. This type of cell death which appears relatively late during the development of some diseases and is considered to result from toxins produced by invading pathogen. But certain mutants were shown to develop cell death associated disease symptoms in the absence of pathogen. HR is activated following perception of attempted infection by pathogens. In addition to the induction of PCD, HR constitutes a coordinated plant response to pathogen attack, which involves: a. oxidative burst, b. nitrosative burst, c. biosynthesis of phytoalexins, d. strengthening the cell walls, e. local and systemic signals for defense reactions in near and distant cells, respectively. Phytotoxins that were considered as simply causing damage to the attacker’s cellular components or as inhibitors of metabolic pathways were recently shown to function as inducers of an active PCD response (Navarre and Wolpert, 1999). Toxins that are secreted by phytopathogenic fungi were found to induce PCD in addition to their inhibitory activity of the host metabolism (Stone et al., 2000). Production of phytoalexins that are low molecular weight secondary metabolites is one of the best defense responses in plants. The specificity of this compound changes depending on the compounds and on the pathogens (Dixon et al., 1994). Consequently, the observed localization of phytoalexin biosynthesis to the area challenged by pathogens corresponds with the induction of PCD in the same cells (Dorey et al., 1997). Phytoalexins are stable compounds and stay in an active form even after the plant cell die. The nature of the PCD inducing signals, offer the possibility to control the PCD response. Several major signal transduction pathways are initiated immediately

after the pathogen perception. These include calcium influx, protein phosphorilation, activation of phospholipases and G proteins. These primary signals are further propagated by the activity of phosphoinositides and G-proteins. These secondary signals lead to the activation of NADPH oxidase. Furthermore, ROS, in turn, possesses multiple signaling activities that induce defense reactions on one hand and PCD on the other hand (Piffanelli et al., 1999; Hancock et al., 2001). Recognition of the pathogen avirulence (Avr) gene products by the plant initiates a signal transduction cascade that activates the HR. The final stage of the HR is PCD that play central role in the disease resistance. Critical steps in the HR are: 1. Interaction of the Avr-gene (X1, X2, X3) with the Resistance gene (R-gene) (RX1, RX2, RX3), 2. Convergence of the signals from the individual R genes into a conserved HR pathway; 3. Activation of NADPH oxidase induces the PCD. The signaling downstream of the NADPH oxidase is regular to almost all types of plant PCD, including developmental PCD and physiological responses to abiotic stress. Additional signaling molecules such as calcium and salicylic acid (SA) regulate NADPH oxidase activation that transforms the extent PCD and associated defense reactions. Following the recognition of pathogens by plants, which is mediated by plant R gene and pathogen Avrgene interactions, signals need to be transmitted and distributed to compartments involved in defense reactions. Application of protein kinase and/or phosphatase inhibitors indicated that the protein phosphorilation and dephosphorilation are involved in a numerous defense responses. Several protein kinases that participate in the perception of specific induction of defense responses have been identified and cloned. SA is a critical signaling molecule in the disease resistance pathways, including PCD and local and systemic resistance (Delaney et al., 1994). SA accumulates more than 100-fold in the challenged area. Treatment of exogenous SA induces many defense genes, phytoalexins and promotes ROS generation and PCD (Shirasu et al., 1997). Many mutants with altered SA perception and signaling have been isolated. The majority of these mutants show corresponding alteration in disease resistance. Interactions between SA, ROS, nitric oxide (NO),

Apoptosis in plants

jasmonic acid (JA) and ethylene and other signaling molecules further complicate the determination of SAspecific functions. The effects of SA is related with activation of the SA-inducible MAP kinase or interaction with SA-response elements in promoters of defense genes, and its inhibitory effect on mitochondria, emphasize the involvement of SA in diverse signaling pathways within the HR signal transduction. Similar to SA many defense responses are modulated by other plant hormones such as jasmonic acid, ethylene and abscisic acid (ABA) (Dong et al., 1998; Klessig et al., 2000). These conclusions are generally based on the analysis of pathogenesis and PCD in hormone signaling mutants.

Methods to detect programmed cell death Although a detailed understanding of how plant cells die is still largely unknown, recent studies have shown that the apoptotic pathways of the animal and plant kingdoms are morphologically and biochemically similar (Greenberg, 1996; Wang et al., 1996). Specifically, the morphological hallmarks of apoptosis include cytoplasmic shrinkage, nuclear condensation, and membrane blebbing (Earnshaw, 1995); the biochemical events involve calcium influx, exposure of phosphatidylserine, and activation of specific proteases and DNA fragmentation, first to large 50-kb fragments and then to nucleosomal ladders (McConkey and Orrenius, 1994; Wang et al., 1996; O'Brien et al., 1998). All of the above-mentioned phenomena were shown to occur in plant PCD. Also, the stimuli that activate apoptosis are similar in plant and animal cells (O'Brien et al., 1998). Although it should be noted that not all of the events were demonstrated in the same plant system, taken together these results infer a common basic cell death process in plants and animals. Morphologically, PCD, known as apoptosis, is generally characterized by a subset of changes such as chromatin and cytoplasm condensation (Vaux, 1993). Little is known about apoptosis in plants including the morphological changes (Danon et al., 2000). Although some accumulating evidence suggests that some features of plant apoptosis such as nuclear disintegration and chromatin condensation triggered endogenously or environmentally are similar to those in animals (reviewed by Danon et al., 2000; Vaux and Korsmeyer, 1999), other features such as cytoplasm

17

shrinkage, nuclear periphery and the formation of apoptotic bodies have not been universally identified. Generally, it seems that chromatin cleavage is the most characteristic feature of PCD (Gavrieli et al., 1992). There are some in situ detection methods, which are dependent on the labelling and detection of the cleaved fragments. ISEL (in situ end labelling), TUNEL and ISNT (in situ nick translation) are three methods that can be used to label these DNA breaks in various tissues. Klenow fragment of DNA Polymerase I is used in the ISEL method to incorporate labelled nucleotides into the DNA strand breaks which occur during internucleosomal cleavage. TUNEL method uses TdT (terminal deoxynucleotidyl transferase) to end label 3'OH groups exposed during the cleavage process (Gorczyca et al., 1993). Although TUNEL-positive reaction is considered as a good specific criterion of death by PCD in animals (Kressel and Groscurth, 1994), some researchers using plant tissues have reported that sample preparation of histological sectioning including fixation, embedding and sectioning, can cause sufficient nicking of nuclear DNA to produce false TUNEL positivity (Wang et al., 1996). Nevertheless, the TUNEL reaction is more specific to PCD when associated with morphological and time-course data, than other death markers such as fluorescein diacetate (FDA) and Evans Blue. Additionally, the fixation procedure is simpler when using a protoplast or cell culture population and has not been reported to induce false TUNEL positive labelling (Danon et al., 2000). Material for current studies of PCD in plants has been obtained predominantly from two different systems, protoplast cell culture for in vitro studies and histological sectioning for in vivo studies (Stein and Hansen, 1999). On tissue sections, PCD changes can only be detected at the tissue level without detailed description in the individual cells unless ultramicroscopy is used and there exists low sensitivity due to poor penetration without pretreatment. The cell wall autofluoresces, resulting in high background that increases as a result of the pretreatment process (Wang et al., 1996). It makes changes in the cells undergoing PCD difficult to visualize. In addition, it has been reported that section preparation including fixation, embedding and sectioning, can cause sufficient nicking of nuclear DNA to produce false TUNEL positive nuclei.

18


Therefore, sectioning techniques should be used very cautiously in plants (Wang et al., 1996). Most of the current knowledge about the nature of PCD has come from the cell culture systems, because cell culture experiments for PCD are generally sufficient than the histological sections. However, a question arises as to whether PCD observed in vitro also occurs in whole plants (Koukalova et al., 1997)? Also, a hallmark of PCD, DNA laddering in cell culture may be caused by mycoplasma endonucleases (Paddenberg et al., 1996). So, in vivo systems might be more biologically relevant than in vitro systems. Therefore, it is necessary to develop more effective techniques for the detection of in vivo plant PCD, both morphologically and biochemically. Nowadays more brightful genomic and proteomic experiments give us a chance to understand molecular levels of biochemically prooved reactions. Especially proteomic approaches will solve the unknowns in protein level. Originally, to study both forms of cell death, necrosis and apoptosis, cytotoxicity assays were used. Generally plant researchers who try to detect PCD in histological sections use the root tips. The root tip of various plant species is generally one of the most sensitive tissues to various environmental impacts (Katsuhara and Kawasaki, 1996), and has previously been used for studying PCD induced by external abiotic factors (Stein and Hansen, 1999). If the material is going to be originated from in vitro system, protoplast culture are the most common technique in plant PCD experiments. These assays were principally of two types: - Radioactive and non-radioactive assays that measure increases in plasma membrane permeability, since dying cells become leaky. - Colorimetric assays that measure reduction in the metabolic activity of mitochondria; mitochondria in dead cells cannot metabolize dyes, while mitochondria in live cells can. However, as more information on apoptosis became available, researchers realized that both types of cytotoxicity assays vastly underestimated the extent and timing of apoptosis. For instance, early phases of apoptosis do not affect membrane permeability, nor do they alter mitochondrial activity. Although the cytotoxicity assays might be suitable for detecting the later stages of apoptosis, other assays were needed to detect the early events of apoptosis. In concert with increased understanding of the physiological events

that occur during apoptosis, a number of assay methods have been developed for its detection. For example, these assays can measure one of the following apoptotic parameters: - Fragmentation of DNA in populations of cells or in individual cells, in which apoptotic DNA breaks into different length pieces. - Alterations in membrane asymmetry. Phosphatidylserine translocates from the cytoplasmic to the extracellular side of the cell membrane. - Activation of apoptotic caspases. This family of proteases sets off a cascade of events that disable a multitude of cell functions. - Release of cytochrome c and AIF into cytoplasm by mitochondria.

DNA fragmentation or laddering method Apoptosis and cell-mediated cytotoxicity are characterized by cleavage of the genomic DNA into discrete fragments prior to membrane disintegration. Because DNA cleavage is a hallmark for apoptosis, assays, which measure prelytic DNA fragmentation, are especially attractive for the determination of apoptotic cell death. The DNA fragments may be assayed in either of two ways: As “ladders” (with the 180 bp multiples as “rungs” of the ladder) derived from populations of cells: The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an irreversible event that commits the cell to die. In many systems, this DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+ dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA fragments. These DNA fragments reveal, upon agarose gel electrophoresis, a distinctive ladder pattern consisting of multiples of an approximately 180 bp subunit. Radioactive as well as non-radioactive methods to detect and quantify DNA fragmentation in cell populations have been developed. In general, these methods are based on the detection and/or quantification of either low molecular weight (LMW) DNA which is increased in apoptotic cells or high molecular weight (HMW) DNA which is reduced in apoptotic cells. The underlying principle of these methods is that DNA, which has undergone

Apoptosis in plants

extensive double-stranded fragmentation (LMW DNA) may easily be separated from very large, chromosomal length DNA (HMW DNA), e.g., by centrifugation and filtration. For the quantification of DNA fragmentation, most methods involve a step in which the DNA of the cells has to be labeled: Prior to the addition of the cell death-inducing agent or of the effector cells, the (target) cells are incubated either with the [3H]thymidine ([3H]-dT) isotope or the nucleotide analog 5-bromo-2’-deoxyuridine (BrdU). During DNA synthesis (DNA replication) these modified nucleotides are incorporated into the genomic DNA. Subsequently, those labeled cells are incubated with cell death-inducing agents or effector cells and the labeled DNA is either fragmented or retained in the cell nucleus. Further, researchers discovered that proteases were involved in the early stages of apoptosis. The appearance of these caspases sets off a cascade of events that disable a multitude of cell functions. Caspase activation can be analyzed in different ways: - By an in vitro enzyme assay. Activity of a specific caspase, for instance caspase 3, can be determined in cellular lysates by capturing of the caspase and measuring proteolytic cleavage of a suitable substrate (Sgonc et al., 1994). - By detection of cleavage of an in vivo caspase substrate. For instance caspase 3 is activated during early stages. Its substrate PARP (PolyADP-Ribose-Polymerase) and the cleaved fragments can be detected with the anti PARP antibody.

TUNEL assay Extensive DNA degradation is a characteristic event which often occurs in the early stages of apoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono- and oligonucleosomes) as well as single strand breaks (“nicks”) in HMWDNA. Those DNA strand breaks can be detected by enzymatic labeling of the free 3’-OH termini with modified nucleotides (X-dUTP, X = biotin, DIG or fluorescein). Suitable labeling enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyl transferase (end labeling). DNA polymerase I catalyzes the template dependent addition of nucleotides when one strand of

19

a double-stranded DNA molecule is nicked. Theoretically, this reaction (In Situ Nick Translation, ISNT) should detect not only apoptotic DNA, but also the random fragmentation of DNA by multiple endonucleases occurring in cellular necrosis. Terminal deoxynucleotidyl transferases (TdT) is able to label blunt ends of doublestranded DNA breaks independent of a template. The end-labeling method has also been termed TUNEL (TdT-mediated XdUTP nick end labeling). The TUNEL method is more sensitive and faster than the ISNT method. In addition, in early stages cells undergoing apoptosis were preferentially labeled by the TUNEL reaction, whereas necrotic cells were identified by ISNT. Thus, experiments suggest the TUNEL reaction is more specific for apoptosis and the combined use of the TUNEL and nick translation techniques may be helpful to differentiate cellular apoptosis and necrosis (Gold et al., 1994). To allow exogenous enzymes to enter the cell, the plasma membrane has to be permeabilized prior to the enzymatic reaction. To avoid loss of LMW DNA from the permeabilized cells, the cells have to be fixed with formaldehyde or glutaraldehyde before permeabilization. This fixation crosslinks LMW DNA to other cellular constituents and precludes its extraction during the permeabilization step. If free 3’ ends in DNA are labeled with biotin- dUTP or DIGdUTP, the incorporated nucleotides may be detected in a second incubation step with (strept)avidin or an antiDIG antibody. The immunocomplex is easily visible if the (strept)avidin or an anti- DIG antibody is conjugated with a reporter molecule (e.g., fluorescein, AP, POD). In contrast, the use of fluorescein-dUTP to label the DNA strand breaks allows the detection of the incorporated nucleotides directly with a fluorescence microscope or a flow cytometer. Direct labeling with fluorescein-dUTP offers several other advantages. Direct labeling produces less nonspecific background with sensitivity equal to indirect labeling and, thus, is as powerful as the indirect method in detecting apoptosis. Furthermore, the fluorescence may be converted into a colorimetric signal if an antifluorescein antibody conjugated with a reporter enzyme is added to the sample.

Annexin V usage in plant PCD determination (Membran alteration) It has been shown that a number of changes in the cell

20


surface (membrane) markers occur during apoptosis, and any one of which may signal “remove now” to the phagocytes in the animal system. These membrane changes include: - Loss of terminal sialic acid residues from the side chains of cell surface glycoproteins, exposing new sugar residues. - Emergence of surface - Loss of asymmetry in cell membrane phospholipids, altering both the hydrophobicity and charge of the membrane surface In theory, any of these membrane changes could provide an assay for apoptotic cells. In fact, one of them has the alteration in phospholipid distribution. In normal cells, the distribution of phospholipids is asymmetric, with the inner membrane containing anionic phospholipids (such as phosphatidylserine) and the outer membrane having mostly neutral phospholipids. In apoptotic cells, however, the amount of phosphatidylserine (PS) on the outer surface of the membrane increases, exposing PS to the surrounding liquid. Annexin V, a calcium-dependent phospholipidbinding protein, has a high affinity for PS. Although it will not bind to normal living cells, Annexin V will bind to the PS exposed on the surface of apoptotic cells. Thus, Annexin V has proved suitable for detecting apoptotic in animal system. There are many studies which use conjugated Annexin V for plant early PCD detection (O’Brien et al., 1997). When we compare the flow cytometry techniques, propidum iodide (PI) is the most common dye to detect apoptosis with cell cycle status in one cell. The PI, which can only enter into, the nucleus of dead cells and intercalate with nuclear DNA, resulting in red fluorescence under ultraviolet light. It also intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adducts that can be excited at 488 nm with a broad emission centred around 600 nm. Since PI can also bind to doublestranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. The excitation of PI at 488 nm facilitates its use on the benchtop cytometers [PI can also be excited in the U.V. (351364 nm line from the argon laser) which should be considered when performing multicolour analysis on the multibeam cell sorters]. Hoechst33342 (HO342) is another DNA fluorochrome which can enter into both live and dead cells (Darzynkiewicz et al., 1992). Other flow cytometric based methods include the

TUNEL assay, which measures DNA strand breaks and Annexin V binding, which detects relocation of membrane phosphatidyl serine from the intracellular surface to the extracellular surface. More recently, one mechanism, which has consistently been implicated in apoptosis, is CASPASE activity (cysteine proteases), typically caspase-3, which can be detected using fluorogenic substrates. Although there are many choices to determine PCD in plants, still there are some unknowns for plant PCD approaches. Well-determined animal system is key way to understand plant PCD but researchers need to investigate details about molecular basis of PCD. Therefore, new genomic and proteomic techniques to understand this question are remarkable. 2D gel electrophoresis or Yeast 2 hybrid techniques especially try to find other related proteins that are still unknown. Microarray technology is another new approach to understand gene expressions in different conditions. We believe that in a short time, new molecules which identify different stages of cell death will be clarified and begin to use for determination.

References Bayly AC, Roberts RA and Dive C. Mechanism of apoptosis. In: Advences in Molecular and Cell Biology, Vol 20, Mechanism of Cell Toxicity. Bittar EE and Chipman JK (eds), Jain Press, Greenwich. 183-229, 1997. Beers EP and Freeman TB. Proteinase activity during tracheary element differentiation in zinnia mesophyll cultures. Plant Physiol. 113: 873-880, 1997. Bell PR. Megaspore abortion: a consequence of selective apoptosis? Int. J. Plant Sci. 157: 1-7, 1996. Blank A and McKeon T.A. Sinlge-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. P roc. Natl Acad. Sci. USA, 86: 3169-3173, 1989. Bozhkov PV, Filonova LH, Suarez MF, Helmersson A, Smertenko AP, Zhivotovsky B and von Arnold S. VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death and Differatiation. 11: 175-182, 2004. Brown PH and Ho TD. Biochemical properties and hormonal regulation of barley nuclease. Eur J. Biochem. 168: 357-364, 1987. Danon A, Delorme VG, Mailhac N and Gallois P. Plant programmed cell death: A common way to die. Plant Physiology and Biochemistry. 38: 647–655, 2000.

Apoptosis in plants Darzynkiewicz Z, Bruno S, DelBino G, Gorczyca W, Hotz MA, Lassota P and Targanos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 13: 795808, 1992. Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negrotto D, Gaffney T, Gutrella M, Kessmann H, Ward E and Ryals J. A central role of salicylic acid in plantdisease resistance. Science. 266: 1247-1250, 1994. Depreatere V and Golstein P. Dismantling in cell death: Molecular mechanisms and relationship to caspase activation. Scand. J. Immunol. 47: 523-531, 1998. Dixon RA, Harisson MJ and Lamb CJ. Early events in the activation of plant defense responses. Annu. Rev. Phytopathol. 32: 479-501, 1994. Dong YH, Zhan XC, Kvarnheden A, Atkinson RG, Morris BA and Gardner RC. Expression of a cDNA from apple encoding a homologue of DAD1, an inhbitor of programmed cell death. Plant. Sci. 139: 165-174, 1998. Dorey S, Baillieul F, Pierrel MA, Saindrenan P, Friting B and Kauffmann S. Spatial and temporal induction of cell death, defense genes, and accumilation of salicylic acid in tobacco leaves reacting hypersensitively to a fungal glycoprotein elicitor. Mol. Plant-Microbe Interact. 10:646-655, 1997. Earnshaw WC. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7: 337-343, 1995. Ellis RE and Horvitz RH. Genetic control of programmed cell death in the nematode C. Elegans. Cell. 44: 817-829, 1986. Evans DE. Programmed cell death in response to abiotic stress. In P rogrammed Cell Death In Plants, John G (Eds), Blackwell Publishing, UK. 194-210, 2004. Fath A, Bethke P, Lonsdale J, Meza-Romero R and Jones R. Programmed cell death in cereal aleurone. Plant Mol. Biol. 44: 255-266, 2000. Fukuda H, and Komamine A. Establishment of an experimental system for tracheary element differentiation from single cells isolated from meshopyll of Zinnia elegans. Plant Physiol. 65:57-60, 1980a. Fukuda H, and Komamine A. Direct evidence for cytodifferantiation to tracheary elements without intervening mitosis in a culture of single cells isolated from meshopyll of Zinnia elegans. Plant Physiol. 65:6164, 1980b. Fukuda H. Xylogenesis: Initiation, progression and cell death. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 299-325, 1996. Fukuda H. Programmed cell death of tracheary elements as a paradigm in plants. Plant Mol. Biol. 44: 245-253, 2000. Gavrieli Y, Sherman Y and Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J. Cell Biol. 119: 493–501, 1992. Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV and Lassmann H. Differentiation between

21

cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab. Invest. 71: 219, 1994. Goodman RN and Novacky AJ. The hypersensitive response reaction in plants to pathogens, a resistance phenomenon. St. Paul, Minnesota, American Phytopathological Society Press. 1994. Gorczyca W, Gong J and Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53: 1945–1951, 1993. Greenberg JT. Programmed cell death: a way of life for plants. P roc. Natl. Acad. Sci. USA. 93:12094-12097, 1996. Greenberg JT. Programmed cell death in plant-pathogen interactions. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48: 525-545, 1997. Groover A, DeWitt N, Heidel A and Jones A. Programmed cell death of plant tracheary elements differentiating in vitro. P rotoplasma. 196: 197-211, 1997. Gunawardena A, Pearce DME, Jackson MB, Hawes CR and Evans DE. Rapid changes in cell wall pectic polysaccharides are closely associated with early stages of aerenchyma formation, a spatial localized form of programmed cell death in roots of maize (Zea mays L.) promoted by ethylene. Plant Cell Environ. 24: 13691375, 2001. Hajouj T, Michelis R and Gepstein S. Clonning and characterization of a receptor-like protein kinase gene associated with senescence. Plant Physiol. 124(3): 13051314, 2000. Hancock JT, Desikan R and Neill SJ. Does the redox status of cytochrome c act as a fail-safe mechanism in the regulation of programmed cell death? F ree Radic. Biol Med. 31: 697-703, 2001. He SY, Bauer DW, Collmer A and Beer SV. Hypersensitive response elicid by Erwinia amylovora harpin requires active plant methabolism. Mol. Plant Micro Inter. 7: 289-292, 1994. Hensel LL, Grbic V, Baumgarten DA and Bleecker AB. Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in arabidopsis. Plant Cell. 5(5): 553-64, 1993. Jackson MB, Fenning TM, Drew MC and Saker LR. Stimulation of ethylene production and gas-space (aerechyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen. Planta. 165: 486-492, 1985. Jones AM and Dangl JL. Logjam at Styx: programmed cell death in plants. Trends Plant Sci. 1: 114-119, 1996. Justin S and Armstrong W. The anatomical characteristics of roots and plant responses to soil flooding. New Phytol. 105: 465-495, 1987. Kao TH and McCubbin AG. How flowering plants discriminate between self and non-self pollen to prevent

22


inbreeding. P roc. Nat Acad. Sci. USA. 93: 12059–12065, 1996. Kaplan DR. Alternative modes of organogenesis in higher plants. In: Contemporary problems in plant anatomy. White RA and Dickson WC (eds): Academic Press, 261300, 1984. Katsuhara M and Kawasaki T. Salt stress induced nuclear and DNA degradation in meristematic cells of barley roots. Plant Cell Physiol. 37: 169–173, 1996. Klessig DF, Durner J, Noad R, Navarre DA, Wendehenne D, Kumar D, Zhou J, Shah J, Zhang S, Kachroo P, Trifa Y, Pontier D, Lam E and Silva H. Nitric oxide and salicylic acid signaling in plant defense. P roc. Natl. Acad. Sci. U.S.A. 97: 8849-8855, 2000. Korsmeyer SJ. Regulation of cell death. Trends Genet. 11: 101-105, 1995. Koukalova B, Kovarik A, Fajkus J and Siroky J. Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress. FEBS Lett. 414: 289–292, 1997. Kressel M and Groscurth P. Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA. Cell and Tissue Research. 278: 549–556, 1994. Kroemer G, Dallaporta B and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Ann. Rev. Physiol. 60: 619-642, 1998. Lai V and Srivastava LM. Nuclear changes during differentiation of xylem vessel elements. Cytobiologie. 12: 220-243, 1976. Lohman KN, Gan S, John MC and Amasino RM. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant. 92: 322-328, 1994. McConkey DJ and Orrenius S. Signal transduction pathways to apoptosis. Trends Cell Biol. 4: 370-375, 1994. Minami A and Fukuda H. Transient and specific expression of a cysteine endopeptidase associated with autolysis during differantiation of Zinnia mesophyll cells into tracheary elements. Plant Cell Physiol. 36: 1599-1606, 1995. Mittler R and Lam E. Sacrifice in the face of foes: Pathogeninduced programmed cell death in higher plants. Trends Microbiol. 4: 10-15, 1996. Navarre DA and Wolpert TJ. Victorin induction of an apoptotic/senescence–like response in oats. Plant Cell. 11: 237-249, 1999. Noodén LD and Guiamét JJ. Genetic control of senescence and aging in plants. In: Handbook of the biology of aging. Academicpress. 94-118, 1996. Noodén LD. Phenomena of senescence and aging. In: Senecence and Aging in plants. Noodén LD and Leopold AC (eds). Academic Press, San Diego. 2-50, 1988. Obara K and Fukuda H. Programmed cell death in xylem differentiation. In: P rogrammed cell death in plants. J. Gray (eds.), Sheffield Academic Press, Sheffield. 131154, 2003.

Obara K, Kuriyama H and Fukuda H. Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia. Plant Physiol. 125: 615-626, 2001. Oberhammer F, Hochegger K, Froschl G, Tiefenbacher R and Pavelka M. Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. J. Cell Biol. 126: 827-837, 1993. O'Brien IEW, Reutelingsperger CPM and Holdaway KM. Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants. Cytometry. 29: 28-33, 1997. O'Brien, IEW, Baguley BC, Murray BG, Morris BAM and Ferguson IB. Early stages of the apoptotic pathway in plant cells are reversible. Plant J. 13: 803-814, 1998. Orzaez D and Granell A. DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum. Plant J. 11:137-144, 1997a. Orzaez D and Granell A. The plant homologue of the defendeer against apoptotic death gene is downregulated during senescence of flower petals. FEBS Lett. 404: 275-278, 1997b. Paddenberg R, Wulf S, Weber A, Heimann P, Beck LA and Mannherz HG. Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. Eur. J. Cell Biol. 71: 105–119, 1996. Piffanelli P, Devoto A and Schulze-Lefert P. Defense signaling pathway in cereals. Curr. Opin. Plant Biol. 2: 295-300, 1999. Raff MC. Social control on cell survival and cell death. Nature. 356: 397-400, 1992. Robatzek S and Somssich IE. Targets of At WRKY6 regulation during plant senescence and pathogen defense. Genes Dev. 16 (9): 1139-1149, 2002. Rouet-Mayer MA, Bureau JM. and Lauriere C. Identification and characterization of lipoxygenase isoforms in senescing carnation petals. Plant Physiol. 98: 971–978,1992. Schussler EE and Longstreth DJ. Aerenchyma develops by cells lysis in roots and cell seperation in leaf petioles in Sagittaria lancifolia (Alismataceae). Am. J. Bot. 83:1266-1273, 1996. Schussler EE and Longstreth DJ. Changes in cell structure during the formation of root aerechyma in Sagittaria lancifolia (Alismataceae). Am. J. Bot. 87:12-19, 2000. Schwartzman, RA and Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocrine Revs. 14: 133-151, 1993. Sgonc R, Boeck G, Dietrich H, Gruber J, Recheis H and Wick G. Simultaneous determination of cell surface antigens and apoptosis. Trends Genet. 10: 41-42, 1994. Shirasu K, Nakajima H, Rajasekhar VK, Dixon RA and Lamb C. Salicylic acid potentiates an agonist-dependent

Apoptosis in plants gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell. 9: 261270, 1997. Stein JC and Hansen G. Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiol. 121: 71–80, 1999. Stone JM, Heard JE, Asai T and Ausubel FM. Stimulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants. Plant Cell. 12:1811-1822, 2000. Studzinski GP. Overview of apoptosis. In: Apoptosis A Practical Approach. Studzinski GP (eds), Oxford University Press, New York. 1-17, 1999. Thelen MP and Northcote DH. Identification and purification of a nuclease from Zinnia elegans L. a potential marker for xylogenesis. Planta. 179: 181-195, 1989. Uren AG, O’Rourke K, Aravind L, Pisabarro MT, Seshagiri Si Koonin EV and Dixit VM. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol. Cell. 6: 961-967, 2000. Vaux DL. Toward an understanding of the molecular mechanisms of physiological cell death. Proc. Natl. Acad. Sci. USA. 90: 786–789, 1993. Vaux DL and Korsmeyer SJ. Cell death in development. Cell. 96: 245–254, 1999. Wang H, Li J, Bostock RM and Gilchrist DG. Apoptosis: a functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. Plant Cell. 8: 375-391, 1996. Willie AH. Cell death. In: Apoptosis and cell proliferation. 2nd edition. Boehringer, Mannheim. VI-VII, 1998. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 284: 555-556, 1980. Yamamato R, Fujioka S, Demura T, Takatsuto S, Yoshida S and Fukuda H. Brassinostreoid levels increase drastically prior to morphogenesis of tracheary elements. Plant Physiol. 125: 556-563, 2001. Yang SH, Berberich T, Sano H and Kusaano T. Specific assotiation of transcripts of tbzF and tbz17, tomacco genes encoding basic region lucine zipper-type transcriptional activators, with guard cells of senecing leaves and/or flowers. Plant Physiol. 127: 23-32, 2001. Yen C and Yang C. Evidence for programmed cell death during leaf senescence in plants. Plant Cell Physiol. 39:922-927, 1998. Zentgraf U and Kolb D. Analysis of differential gene expression during leaf senescence using Arabidopsis high density genome arrays. 13 Conress FESPP, 2002.

23