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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
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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
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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
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Narcin Palavan-Unsal et al.
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
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Narcin Palavan-Unsal et al.
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
Narcin Palavan-Unsal et al.
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
Narcin Palavan-Unsal et al.
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
Narcin Palavan-Unsal et al.
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.
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