Programmed cell death

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1. Details of Module and its Structure

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Module Detail

Subject Name <BOTANY>

Paper Name <Cell Biology>

Module Name/Title <programmed cell death>

Module Id <>

Pre-requisites Basic knowledge about plant cell.

Objectives To make the students aware of the programmed cell death and its regulation and mechanism.

Keywords Programmed cell death, apoptosis, autophagy

Structure of Module / Syllabus of a module (Define Topic / Sub-topic of module )

<PCD > <Sub-topic Name1>, <Sub-topic Name2>

<Topic name2> <Sub-topic Name2.1>, <Sub-topic Name2.2>

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2. Development Team

Programmed cell death

Contents

 1. History

 2. Types

o 2.1 Apoptosis o 2.2 Autophagy o 2.3 Other types

 3 Atrophic factors

 4. Morphological and physiological events in a cell

 5. Molecular mechanis

o 5.1 Three Classes of Proteins Function in the Apoptotic Pathway o 5.2 Pro-Apoptotic Regulators Promote Caspase Activation

o 5.3 Some Trophic Factors Prevent Apoptosis by Inducing Inactivation of a Pro- Apoptotic Regulator

 6. Programmed cell death in response to abiotic stress

 7. Programmed cell death in response to biotic stress

Role Name Affiliation

National Coordinator <NA>

Subject Coordinator <Dr. Sujata Bhargava>

Paper Coordinator <Dr. Nutan Malpathak>

Content Writer/Author (CW) <Dr. Amrita Srivastav >

Content Reviewer (CR) < Dr. Nutan Malpathak >

Language Editor (LE) < Dr Laate >

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Introduction:

All organisms are made up of cells, which are the structural unit of living beings. There is a particular system through which the cell starts its cycle and ends it. When a cell ends its life cycle, it’s through a particular systematic Program, which is known as programmed cell-death (or PCD).

This is run through the intercellar program of the cell and is a highly regulated process. It is a condition in which the cell undergoes suicidal mechanism.

Some common examples of PCD can be seen in the developing human embryo. We are aware of the fact that “ontogeny replicates phylogeny”. The embryo of humans looks the same as that of any other vertebrate but for the formation of fingers in human babies the cells between the fingers and toes undergo Programmed cell death , and due to this phenomenon the digits separate giving rise to 5 fingers and 5 toes.

1. History

The concept of PCD came into existence as Lockshin & Williams in 1964 used it in the explanation to study insect tissue development.

The mechanism of PCD was first understood by the studies of Apoptosis regulator Bcl-2 which is from a family of evolutionarily related proteins. BCL2,is protein from an oncogene which is activated by chromosome translocations from follicular lymphoma. BCL2 caused cancer by inhibiting lymphoma cells from killing themselves. These proteins govern mitochondrial outer membrane permeabilization (MOMP) and can be either pro-apoptotic (Bax, BAD, Bak and Bok among others) or anti-apoptotic (including Bcl-2 proper, Bcl-xL, and Bcl-w, among an assortment of others). There are a total of 25 genes in the Bcl-2 family known to date.

2. Types

Apoptosis also known as Type I cell-death and autophagy i.e Type II cell-death are the two types of programmed cell death known.

2.1 Apoptosis

It is a kind of programmed cell death that occurs in multicellular organisms. A lot of biochemical alterations occur in the cell leading to changes in its morphology and finally death of the cell.

Blebbing, cell shrinkage, fragmentation of the nucleus, condensation of the chromatin along with fragmentation of chromosomal DNA are seen as morphological changes in the cell. Certain survival factors help a cell to live if they become absent they would provide an instinct for suicide to the cells. Some evidences also show that endonuclease activation can be a reason for apoptosis,however, presumably true apoptosis and programmed cell death must be genetically mediated.

It is also evident that mitosis and apoptosis are interlinked and that the balance achieved depends on signals received from certain growth or survival factors in the organisms.

2.2 Autophagy

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Autophagy or Macroautophagy, is a breakdown process which results in the autophagosomic- lysosomal degradation of bulky cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles.

Autophagy is generally activated when the cell is under nutrient deficiency but has also been associated with physiological and pathological processes like development, differentiation, neurodegenerative diseases, stress, infection and cancer.

2.3 Other types

Necroptosis or caspase-independent programmed cell-death or even called non-apoptotic programmed cell-death is another pathway that has been discovered as programmed cell death which many times work as backup mechanisms for PCD.

Other forms of programmed cell death include anoikis, which is identical to apoptosis except in its induction; cornification, a kind of cell death exclusive for the eyes; excitotoxicity; ferroptosis, an iron-dependent form of cell death and Wallerian degeneration.

3. Atrophic factors

An atrophic factor is a natural force that compels a cell to die.^] Common types of atrophic factors are:

1. Decreased workload 2. Loss of innervation 3. Less blood supply 4. nutrient deficit

5. Loss of endocrine stimulation 6. Senility

7. Compression

4. Morphological and physiological events in a cell

Structurally apoptotic cells are small condensed bodies. The dense and fragmented chromatin is packed into compact membrane-bound bodies with scattered cell organelles. The plasma membrane loses its specific architecture and exhibits high blebbing. It cuts off projections so that one complete cell may split into various membrane-bound apoptotic bodies. The plasma membrane also shows significant chemical changes so that the apoptotic bodies are recognized by the phagocytes.

Macromolecular synthesis is an essential pre-requisite for induction of programmed death in cells.

The endonuclease cuts the double-stranded linker DNA between nucleosomes at regular inter- nucleosome sites. Zinc acts as an inhibitor of endonuclease whereas Calcium activates the enzyme.

Research has shown that signal transduction controls the Ca2+ flux in the cells via the receptors.

Synthesis of RNA and proteins is also required in the apoptotic cells. High levels of mRNA are for

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several degradative enzymes in the suicidal cells. Various regulatory proteins maintain a control over the regulatory steps of PCD.

At the molecular level, mammalian equivalents of the cell death (ced) gene are being found.

Presence of many specific genes at PCD level is being indicated. Specific expression of cell death- associated gene products (e.g. TRPM-2/SGP-2) has been reported in several unrelated apoptotic cell systems. Sequential induction of c-fos, c-myc and 70 kDa heat shock protein is reported. Studies show that particular genes should remain in transcriptionally active demethylated state while programmed cell death. Recent studies demonstrate that apoptosis is positively or negatively governed by specific genes.

5. Molecular mechanism of programmed cell death

Plant growth and development includes the degradation of cell organelles, protoplasts, different tissues and organs. It is a phenomenon to eliminate redundant, misplaced or damaged cells and maintain multicellular organisms.

Apoptosis and plant programmed cell death have similarities like DNA laddering, caspase-like proteolytic activity in the cells and cytochrome c release from the mitochondria.

Programmed cell death or apoptosis has an important role in animal development, metamorphosis, and tissue homeostasis. It is a genetically controlled physiological process that has two distinct and sequential processes: the death of cells, and their subsequent removal by engulfing cells.

5.1 A Cascade of Caspase Proteins

Key insights into the molecular mechanism regulating cell death came from genetic studies in C.elegans. Of the 947 nongonadal cells generated during development of the adult hermaphrodite form, 131 cells undergo programmed cell death. Specific mutations have identified a variety of genes whose encoded proteins play an essential role in controlling PCD during C. elegans development. For instance, a PCD not occur in worms carrying loss of functions mutations in the ced-3 gene or the ced-4 gene, as a result the 131 “doomed”

cells survive.CED-4 is a protease-activating factor that causes autocleavage of the CED-3 precursor protein creating an active CED-3 protease that initiates cell death. In contrast , in ced-9 mutants, all die during embryonic life, so the adult form never develops.

Three Classes of Proteins Function in the Apoptotic Pathway

Key insights into the molecular mechanisms regulating cell death came from genetic studies in C.

elegans. Scientists have traced the lineage of all the somatic cells in C. elegans from the fertilized egg to the mature worm simply by following thedevelopment of live worms under Nomarski optics.

Of the 1090 somatic cells generated during development, 131 cells undergo programmed cell death.

Specific mutations have identified a variety of genes whose encoded proteins play an essential role

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in controlling this process. For instance, in worms carrying mutations in the ced-3 or the ced- 4 genes, programmed cell death does not occur, and all 1090 cells survive. In contrast, in ced- 9 mutant animals, all 1090 cells die. These genetic studies indicate that the CED-3 and CED-4 proteins are required for cell death, that CED-9 suppresses apoptosis, and that the apoptotic pathway can be activated in all cells. Moreover, the finding that cell death does not occur in ced- 9/ced-3 double mutants suggests that CED-9 acts upstream of CED-3 to suppress the apoptotic pathway.

That apoptosis involved an evolutionarily conserved pathway was first suggested by the confluence of genetic studies in worms and studies on human cancer cells (Figure 23-49). The first apoptotic gene to be cloned, bcl-2, was isolated as a breakpoint rearrangement in human follicular lymphomas and was shown to act as an oncogene that promoted cell survival rather than cell proliferation. Not only are the Bcl-2 and CED-9 proteins homologous, but a bcl-2 transgene can block the extensive cell death found in ced-9 mutant worms. Thus both proteins act as regulators that suppress the apoptotic pathway. In addition, both proteins contain a single transmembrane domain and are localized to the outer mitochondrial, nuclear, and endoplasmic

reticulum membranes.

Fig 1. Overview of the apoptotic pathway in C. elegans and vertebrates

Three general types of proteins are critical in this conserved pathway. Regulators either promote or suppress apoptosis; the two regulators shown here, CED-9 and Bcl-2, both function to suppress apoptosis in the presence of trophic factors. Adapters interact with both regulators and effectors; in the absence of trophic factors, they promote activation of effectors. A family of cysteine proteases serve as effector proteins; their activation leads to degradation of various intracellular substrates and eventually cell death. [Adapted from D. L. Vaux and S. J. Korsemeyer, 1999, Cell 96:245.]

The effector molecules in the apoptotic pathway are a family of enzymes called caspases, so named because they are cysteine proteases that selectively cleave proteins at sites just C-terminal to aspartate residues. These proteases have specific intracellular targets such as proteins of the nuclear lamina and cytoskeleton. Cleavage of these substrates leads to the demise of a cell. Activation of caspases, discussed below, appears to be a common feature of most, if not all, cell-death programs.

The principal effector protease in C. elegans is CED-3. Mammalian cells contain multiple caspases.

Cell-culture studies have yielded important insights into how the various CED proteins in C.

elegans and the related mammalian proteins act together to control apoptosis. Expression of C.

elegans CED-4 in a human kidney cell line induces rapid apoptosis. This can be blocked by co- expression of CED-9 (or mammalian Bcl-2). CED-9 directly binds to CED-4 and relocalizes it from the cytosol to intracellular membranes. Thus the pro-apoptotic function of CED-4 is directly

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suppressed by the anti-apoptotic function of CED-9. CED-4 also binds directly to CED-3 (and related mammalian caspases) and promotes activation of its protease activity. Biochemical studies have shown that CED-4 can simultaneously bind both to CED-9 and CED-3.

5.2 Pro-Apoptotic Regulators Promote Caspase Activation

Having introduced the major participants in the apoptotic pathway, we now take a closer look at how the effector caspases are activated in mammalian cells. Although CED-9 and Bcl-2 suppress the cell-death pathway, other regulatory proteins act to promote apoptosis. The first pro-apoptotic regulator to be identified, named Bax, was found associated with Bcl-2 in extracts of cells expressing high levels of Bcl-2. Sequence analysis demonstrated that Bax is related in sequence to CED-9 and Bcl-2, but overexpression of Bax induces cell death rather than protecting cells from apoptosis, as CED-9 and Bcl-2 do. Thus this family of homologous regulatory proteins comprises both anti-apoptotic members (e.g., CED-9, Bcl-2) and pro- apoptotic members (e. g., Bax). All members of this family, which we refer to as the Bcl-2 family, are single-pass transmembrane proteins and can participate in oligomeric interactions.

Thus the fate of a given cell—survival or death—may reflect the particular spectrum of Bcl-2 family members present in the cell and the intracellular signaling pathways regulating them.

Recent studies suggest that Bcl-2 family members can influence the subcellular distribution of cytochrome c; moreover, biochemical studies have implicated cytochrome c in caspase activation. The current model of caspase activation in mammalian cells, summarized in Figure 23-50a, accounts for the involvement of cytochrome c. In normal healthy cells, cytochrome c is localized between the inner and outer mitochrodrial membrane, but in cells undergoing apoptosis, cytochromec is released into the cytosol. This release can be blocked by overexpression of Bcl-2; conversely, overexpression of Bax promotes release of cytochrome c into the cytosol and apoptosis. In the cytosol, binding of cytochrome c to the adapterprotein Apaf-1 (i.e., mammalian CED-4) promotes activation of a caspase cascade. Bax homodimers, but not Bcl-2 homodimers or Bcl-2/Bax heterodimers, permit influx of ions through the mitochondrial membrane. It remains unclear how this ion influx acts to trigger the release of cytochrome c.

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Figure 2 Current models of the intracellular pathways leading to cell death by apoptosis or to trophic factor–mediated cell survival in mammalian cells

The details of these pathways in any given cell type are not yet known. (a) In the absence of a trophic factor. Bad, a soluble pro-apoptotic protein, binds to the anti-apoptotic proteins Bcl-2 and Bcl-xl, which are inserted into the mitochondrial membrane. Bad binding prevents the anti- apoptotic proteins from interacting with Bax, a membrane-bound pro-apoptotic protein. As a consequence, Bax forms homo-oligomeric channels in the membrane that mediate ion flux.

Through an as-yet unknown mechanism, this leads to the release of cytochrome c from the space between the inner and outer mitochondrial membrane. Cytochrome cthen binds to the adapter protein Apaf-1, which in turn promotes a caspase cascade leading to cell death. (b) In the presence of a trophic factor such as NGF. In some cells, binding of trophic factors stimulates PI-

3 kinase activity, leading to activation of the downstream kinase Akt, which phosphorylates Bad.

Phosphorylated Bad then forms a complex with the 14 - 3 - 3 protein. With Bad sequestered in the cytosol, the antiapoptotic Bcl-2/Bcl-xl proteins can inhibit the activity of Bax, thereby preventing the release of cytochrome c and activation of the caspase cascade. [Adapted from B.

Pettman and C. E. Henderson, 1998, Neuron 20:633.]

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Fig. 3 Some Trophic Factors Prevent Apoptosis by Inducing Inactivation of a Pro-Apoptotic Regulator

We saw earlier that neutrophins such as nerve growth factor (NGF) protect neurons from cell death.

The intracellular signaling mechanisms linking such survival factors to inactivation of the cell-death machinery are not known in detail, but some intriguing clues are available. The finding that trophic factors appear to work largely independent of protein synthesis suggested that the activity of one or more components of the cell-death pathway is altered post-translationally in response to

intracellular signals activated by binding of trophic factors to their receptors. Scientists demonstrated that in the absence of trophic factors, the nonphosphorylated form of Bad is associated with Bcl-2/Bcl-xl at the mitochondrial membrane. Binding of Bad inhibits the anti- apoptotic function of Bcl-2/Bcl-xl, thereby promoting cell death. Phosphorylated Bad, however, cannot bind to Bcl-2/Bcl-xl and is found in the cytosol complexed to the phosphoserine-binding protein 14-3-3. Hence, signaling pathways leading to Bad phosphorylation would be particularly attractive candidates for transmitting survival signals.

A number of trophic factors including NGF have been shown to activate PI-3 kinase, which in turn activates a downstreamkinase called Akt. This kinase phosphorylates Bad at sites known to inhibit its pro-apoptotic activity. Moreover, a constitutively active form of Akt can rescue cultured

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neutrophin-deprived neurons, which otherwise would undergo apoptosis and die. These findings support the mechanism for the survival action of trophic factors depicted in Figure2b. In other cell types, different trophic factors may promote cell survival through post-translational modification of other components of the cell-death machinery.

6. Programmed 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 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 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

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(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..

7. Programmed cell death in response to biotic stress

Many studies have demonstrated the induction of PCD in plants in response to pathogen attack, indicating that 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 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:

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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), 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.

SUMMARY

 Cells die by murder or suicide through programmed cell death, often referred to as apoptosis.

 All cells require trophic factors to prevent apoptosis and thus survive.

 The best-characterized trophic factors are the neutrophins, including NGF, BDNF, and NT- 3. During development, neurons compete for a limited supply of these trophic factors in their target fields. As a result, many cells undergo programmed cell death, so that the number of surviving neurons matches the target-field size.

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 Genetic studies in C. elegans defined an evolutionarily conserved cell-death pathway with three major components (see Figure 1). The C. elegans anti-apoptotic protein, CED-9, is structurally and functionally homologous to Bcl-2 in vertebrates.

 The effectors of cell death are cysteine proteases, called caspases. Once activated, these proteases cleave specific intracellular substrates leading to the demise of a cell. Pro- apoptotic proteins promote caspase activation, and anti-apoptotic proteins suppress activation.

 Direct interactions between pro-apoptotic and anti-apoptotic proteins lead to cell death in the absence of trophic factors. Binding of extracellular trophic factors can trigger modulation of these interactions via phosphorylation or other post-translational modifications, resulting in cell survival

Apoptosis

Contents:

1. Introduction

2. Programmed celldeath occurs through Apoptosis 3. Occurrence of apoptosis in plants

3.1 Apoptosis in vegetative plant tissues 3.2 Xylogenesis

3.3 Apoptosis during reproductive period 3.4 Apoptosis in senescence

3.5 Apoptosis in pollen prevents inbreeding 4. Detection methods of apoptosis

5. Summary

1. Introduction:

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 PCD and more commonly Apoptosis.

The term apoptosis comes from plant kingdom from old Greek apoptosis that

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originally means the loss of petals or leaves. Apoptosis occurs in developing vertebrate nervous system and adult animal system. In the developing vertebrate nervous 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 through the process of Apoptosis.

2. Programmed cell-death occurs through Apoptosis

A molecular mechanism for eliminatingdevelopmentally unwanted cells is essential forsuccessful development and growth of complexmulticellular organisms. Therefore in addition toregulating the rate of cell division, multicellularorganisms such as animals and plants contain abiochemical pathway to control cell death.

Bycoordinating the activation of cell division and celldeath, animals and plants may direct a variety ofdevelopmental processes such as generation ofdevelopmental patterns and the shaping of cells,tissues and organs. However, cell death may not belimited to development and may also be used in anumber of other processes such as control of cellpopulations and defense against invading microbes.

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

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Fig 1 : Apoptosis shaping individuals

Table: Differences between apoptosis and necrosis.

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3. Occurrence of apoptosis in plants

Plants eliminate cells, organs and parts duringresponses to stress and expression during variousdevelopmental processes:

3.1 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.

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.

3.2 Xylogenesis

The most important 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. Recent progress in the study of tracheary elements PCD has been made mainly with an in vitro Zinniasystem established by Fukuda and Komamine in 1980s. In this system single mesophyll cells isolated from Zinnia leaves transdifferentiate synchronously into tracheary elements at a high frequency without cell division.

A number of ultrastructural observations of the PCD in tracheal element

differentiation have been reported in 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. Nucleoids in chloroplasts are also degraded rapidly after vacuole

rupture. Cytoplasmic streaming ceases immediately after the disruption ofthe

vacuole. All these observations revealed that one of the most critical steps in PCD is

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the irreversible disruption of tonoplast. Secondary wall lignification is initiated before the vacuole rupture. It was found recently thatbrassinosteroid biosynthetic pathway is activated before the tracheary element PCD, and the synthesized brassinosteroids induce PCD and the formation of secondary cell walls.

Nuclear shrinkage and fragmentation do not occur in tracheary PCD, no prominent chromatin condensation is established, although the nucleus sometimes exhibitschromatin condensation near the nuclear envelope.Cellular shrinkage, membrane blebbingand 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 bandswere detected by gel assay. 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. Extracts from

Zinnia cells cultured in trachearyelement inductive medium contain cystein protease

activity. 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. Much 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.

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Fig. 2 Formation of Plant TrachearyElements

3.3 Apoptosis during reproductive period

The unpollinatedflowers are fully thrown away. Ovarieswith fertilized egg cells in ovules on the same plant areretained forming fruits while the other parts;

petals,sepals or tepals fall off. Stigmas and pistils may alsobe eliminated. In apomictic species, the fruits develop without fertilization, which means that the ovarieswith ovules are retained forming fruit, but the otherflower parts are eliminated.

Apoptosis is involved in the formation of femalegametes in seed plants. Single meiotic division givesfour haploid megaspore cells, three of them undergoapoptosis, remaining one have two additional mitoticdivision and bring to egg and associated cells of theembryo. Apoptosis is also involved in theformation of male sexual organs.

Tapetum layer issurrounding the pollen during maturation undergoes

Apoptosis. Plants developed several mechanisms to avoidself-pollination. One of

these involves inhibition ofgerminating pollen dependent on recognition by

pistiltissue. This process is mediated by proteins showingRNase activity, which is

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crucial for their function. The growth of the pollen tubethrough the pistil is associated by selective cell death.Therefore pistil cells along the growth way of thepollen tube undergo apoptosis while the rest of thetissue stays intact. Two synergidcells are present at the entry to the egg sack, one ofthem undergoes apoptosis for arriving pollen tube toenter and release sperm cells.

Apoptosis also occurs during the embryogenesis inplants. Cell death within the embryo does occur as partof its normal development and includes the death ofscutellar cells surrounding the developing radicle,death of suspensor and death of nucellus from whichthe egg cell originates. These cell types that undergocell death are highly specific and their death isessential for the final development of the embryo. Inaddition, in some species the transient endospermundergoes a cell death that is followed by itsreabsorption during embryogenesis that is thought tofacilitate embryo growth, whereas in other species inwhich the endosperm is persistent, it urvives as a partof the mature seed.Apoptosis occurs during the germination of plantsand it is also formed in the seed storage tissues.Endosperm supplies nutrients to the embryo fordevelopment and germination and undergoes PCD.This process generally associated with lytic enzymeactivities, for instance α-amylase is secreted fromaleurone layer which surrounds the endosperm. Using a model system ofbarley aleurone protoplasts, scientists revealedthat this PCD occurs in a gibberellic acid dependentmanner.

3.4. Apoptosis in senescence

Senescence is a complex, highly ordered process, during whichplant organs undergo a series of biochemical and physiologicalchanges. These developmental changes result in the degradationof macromolecules and the recycling of their components to other parts of the plant. Senescence ends in death of the organ. However, senescence is a process that requires cell viability and is oftenreversible. Theoverlap between senescence and PCD is synchronous.However when senescence-associated PCD was activated it initiated a cascade of eventsthat destroyed the cell over a relatively short period of time(3–12 h) and therefore operated under the same timelines asPCD activated by heat stress in cucumber cotyledons study. This led to the conclusion thatPCD either operated as the final instalment of senescence,or equally plausible, operated following senescence. Van Doorn&Woltering defined PCD as ‘the process thatleads to the moment of death and the degradation that goeson after this moment’.

This broad definition allows them toargue that PCD and senescence are synchronous.

However,based on our apoptotic-like criteria for death we can equallyargue that

senescence and AL-PCD either do not overlap ordo so only transiently. The

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distinction of the extent of overlapof senescence and PCD is not simply a semantic argument, asmany studies have been carried out to identify senescent genesthat may be involved in activating theapoptotic-like cell-deathprocess. In fact, we would argue that the opposite may bethe case and AL-PCD may be suppressed during senescence.Senescent cells are being actively recycled, which means thatthe cells are subjected to nucleases, proteases and photosyntheticbreakdown along with various other senescent-related stresses.

Normally one might expect that a cell which is subjected tothis type of stress would activate PCD. However, in order tocomplete senescent recycling, PCD may have to be activelysuppressed during the senescence process and only activatedwhen recycling has been completed. For example, Baxinhibitor-1 (BI-1) suppresses PCD inboth plant and animal cells.Baxinhibitor-1 is upregulated during flower senescence in bothoilseed rape and tobacco duringharvest-induced senescence in broccoli.

Similarly, another PCD suppressor, defender against apoptoticdeath (DAD1), is upregulated during senescence of leaves,fruits and relatively long-lasting petals in apple, whereas it is downregulated before the onset of apoptotic-like cell death in shorter-lived petals of pea and gladiolus. Senescence may indeed be a rich sourceof AL-PCD genes, but genes involved in suppression, ratherthan activation, of the process,and indeed activation of AL-PCD, may be a result of senescence having terminated andconsequently a cessation of transcription of PCD-suppressinggenes and gene products.

3.5Apoptosis in pollen prevents inbreeding

During pollination, plants enforce self-incompatibility (SI) as an important means to

prevent self-fertilization. Research on the corn poppy (Papaverrhoeas) has revealed

that proteins in the pistil on which the pollen lands, interact with pollen and trigger

PCD in incompatible (i.e., self) pollen. The researchers, Steven G. Thomas and

Veronica E. Franklin-Tong, also found that the response involves rapid inhibition of

pollen-tube growth, followed by PCD.

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Fig 3 Apoptosis is a fundamental process of life. During the evolution of

multicellular organisms, the actively controlled demise of cells has been recruited to fulfil a multitude of functions in development, differentiation, tissue homeostasis, and immune systems. In this review we discuss some of the multiple cases of Apoptosis that occur as integral parts of plant development in a remarkable variety of cell types, tissues, and organs. Although research in the last decade has discovered a number of PCD regulators, mediators, and executers, we are still only beginning to understand the mechanistic complexity that tightly controls preparation, initiation, and execution of PCD as a process that is indispensable for successful vegetative and reproductive development of plants.

4. Detection methods of apoptosis Assays for Apoptosis

Since apoptosis occurs via a complex signaling cascade that is tightly regulated at

multiplepoints, there are many opportunities to evaluate the activity of the proteins

involved. As theactivators, effectors and regulators of this cascade continue to be

elucidated, a large numberof apoptosis assays are devised to detect and count

apoptotic cells. However, many features ofapoptosis and necrosis can overlap, and it

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is therefore crucial to employ two or more distinctassays to confirm that cell death is occurring via apoptosis. One assay may detect early(initiation) apoptotic events and a different assay may target a later (execution) event. Thesecond assay, used to confirm apoptosis, is generally based on a different principle.

Multiplexing, which is the ability to gather more than one set of data from the same sample,is another methodology for apoptosis detection that is becoming increasingly popular. Thereare a large variety of assays available, but each assay has advantages and disadvantages whichmay make it acceptable to use for one application but inappropriate for another application(Watanabe et al., 2002; Otsuki et al., 2003).

Therefore, when choosing methods of apoptosisdetection in cells, tissues or organs, understanding the pros and cons of each assay is crucialUnderstanding the kinetics of cell death in each model system is also critical. Some proteins,such as caspases, are expressed only transiently. Cultured cells undergoing apoptosis in vitrowill eventually undergo secondary necrosis. Apoptotic cells in any system can die and disappear relatively quickly. The time from initiation of apoptosis to completion can occur asquickly as 2–3 hours. Therefore a false negative can occur if the assay is done too early or toolate. Moreover, apoptosis can occur at low frequency or in specific sites within organs, tissuesand cultures. In such cases, the ability to rapidly survey large areas could be useful. In general,if detailed information on the mechanism of cell death is desired, the duration of toxin exposure,the concentration of the test compound and the choice of assay endpoint become critical.

A detailed description of all methodologies and assays for detecting apoptosis is beyond thescope of this article. However, some of the most commonly employed assays are mentionedand briefly described. Apoptosis assays, based on methodology, can be classified into six majorgroups and a subset of the available assays in each group is indicated and briefly discussed:

a.Cytomorphological alterations b.DNA fragmentation

c.Detection of caspases, cleaved substrates, regulators and inhibitors d.Membrane alterations

e.Detection of apoptosis in whole mounts

Summary

 Programmed cell death (PCD) is an integral part of plant life.

 Numerous PCD instances occur during regular plant development.

 Developmental PCD is tightly linked with cellular differentiation.

 Successful vegetative and reproductive development depends on precise PCD control

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