Plant, Cell and Environment (2012) 35, 234–244
Redox regulation in plant programmed cell death
M. C. DE PINTO*1, V. LOCATO*2 & L. DE GARA2
1
Dipartimento di Biologia, Università degli Studi di Bari, via E. Orabona 4, 70125 Bari and 2Centro Integrato di Ricerca,
Università Campus Bio-Medico di Roma, via A. del Portillo 21, 00128 Roma, Italy
ABSTRACT
Programmed cell death (PCD) is a genetically controlled
process described both in eukaryotic and prokaryotic
organisms. Even if it is clear that PCD occurs in plants, in
response to various developmental and environmental
stimuli, the signalling pathways involved in the triggering of
this cell suicide remain to be characterized. In this review,
the main similarities and differences in the players involved
in plant and animal PCD are outlined. Particular attention
is paid to the role of reactive oxygen species (ROS) as key
inducers of PCD in plants. The involvement of different
kinds of ROS, different sites of ROS production, as well as
their interaction with other molecules, is crucial in activat-
ing PCD in response to specific stimuli. Moreover, the
importance is stressed on the balance between ROS pro-
duction and scavenging, in various cell compartments, for
the activation of specific steps in the signalling pathways
triggering this cell suicide process. The review focuses on
the complexity of the interplay between ROS and antioxi-
dant molecules and enzymes in determining the most suit-
able redox environment required for the occurrence of
different forms of PCD.
Key-words: antioxidants; ascorbate; programmed cell death;
reactive oxygen species; redox homeostasis.
INTRODUCTION
The term ‘programmed cell death’ (PCD) appeared in the
scientific literature for the first time, in 1966, to describe a cell
death process controlling tadpole tail development (Tata
1966). In the following years, attention was focused on the
importance of PCD during embryo development; moreover,
it was found that exogenous agents, including anti-cancer
drugs, were able to trigger this cell suicide process (Kerr,
Wyllie & Currie 1972; Sulston 1976; Hedgecock, Sulston &
Thomson 1983).These studies were determinant in awarding
the Nobel prize to Sydney Brenner, John Sulston and Bob
Horvitz in 2002, and attracted research workers’ attention to
Correspondence: L. De Gara. Fax: +39 06 225411966; e-mail:
l.degara@unicampus.it
*These authors have equally contributed to the paper.
234
doi: 10.1111/j.1365-3040.2011.02387.
pce_2387 234..244
the possibility of modulating PCD as a promising therapeu-
tic strategy with broad implications, in particular for cancer
and degenerative diseases (Nicholson 2000).
The possibility of activating PCD in a limited number of
cells is now recognized as a key mechanism in organ
development and in tissue homeostasis. PCD also plays a
relevant role in the defence responses against certain
kinds of environmental stresses. Since the end of 1990s,
PCD has also been observed in unicellular eukaryotes
(Ameisen 1996) and even in bacteria, where it has been
interpreted as a form of ‘altruistic’ controlled self-
destruction, in line with the multicellular behaviour of
colonial protozoa and bacteria (Engelberg-Kulka et al.
2006). In unicellular organisms, PCD also contributes to
select the fittest cells for a given environment (Ameisen
et al. 1995). On this basis, it could be considered as an evo-
lutionary strategy promoting conservation of the genetic
characteristics of unicellular species submitted to a chang-
ing environment.
In multicellular organisms, several forms of PCD have
been described, according to the tissue or organ involved, as
well as the specific stimulus triggering the process. In plants,
many growth and developmental processes require the
induction of PCD, such as development of endosperm and
aleurone cells in cereals or seed storage tissues (Fath et al.
2000; Young & Gallie 2000; Lombardi et al. 2010), differen-
tiation of tracheary elements (Fukuda 2000; Kwon, Cho &
Park 2010), female gametophyte differentiation (Wu &
Cheung 2000), leaf abscission and whole plant senescence
(Gahan 1982; Buckner, Janick-Buckner & Johai 1998; Lee
et al. 2011), to cite only types of PCD which have been
extensively characterized. Plant pathogen attack can also
induce PCD at the infection sites through a mechanism
known as hypersensitive response (HR). This response,
occurring in the incompatible plant–pathogen interaction,
allows the plant to isolate the pathogen in an inhospitable
environment, thus limiting pathogen spread and avoiding
greater damage and even the death of the entire plant
(Greenberg 1996). Another form of ‘altruistic’ PCD occurs
in response to hypoxia and determines the degradation of
certain parenchymatic cells of roots for the formation of
aerenchyma (Drew, He & Morgan 2000). PCD also occurs
as a consequence of several other abiotic forms of stress
(heat stress, ozone, etc.) (Pasqualini et al. 2003; Locato et al.
2008), even if, in these cases, the advantage of PCD is not
always clear.
© 2011 Blackwell Publishing Ltd

Plant versus animal PCD
The term PCD was introduced to underline the active
involvement of cell metabolism in triggering a form of cell
death displaying different features from necrosis, which was
initially considered a passive event of death, due to severe
cell injury (Fietta 2006). Despite new evidence that has led
to a revaluation of this traditional view of necrosis (Hitomi
et al. 2008), PCD remains the most general term used to
indicate an active cell suicide. In the plant field, the term
PCD includes different forms of active cell death, which are
far from being fully characterized. On the contrary, in the
animal field, three different types of PCD have been recog-
nized: apoptosis (I), autophagy (II) and a less characterized
type III that has been mostly indicated as necrosis-like
(reviewed by Portt et al. 2011). Since apoptosis was the first
form of PCD to be discovered and characterized in animal
systems, plant PCD has often been investigated in compari-
son to the best-known animal process. However, plant PCD
is not synonymous of apoptosis since different forms of
plant PCD have been found to exhibit only some morpho-
logical and molecular hallmarks of apoptosis (Danon et al.
2000; Cacas 2010). For instance, in plant PCD, at least in cell
culture models, cell condensation has been observed
(Hiraga et al. 2010; Lytvyn, Yemets & Blume 2010) (Fig. 1).
This condensation is evident as a cytoplasmic shrinkage, a
sort of plasmolytic process, in which the plasma membrane
separates from the cell wall, but it is not accompanied by
cell fragmentation and formation of the so-called cellular
apoptotic bodies, which characterize apoptosis. This distinc-
tive feature is probably due to the presence of the cell
wall and the absence of phagocytes in plants. Nuclear and
chromatin condensation, as well as DNA laddering, are
apoptosis hallmarks also found in several forms of plant
PCD (Fig. 1; Young & Gallie 2000; Vacca et al. 2004; de
Pinto et al. 2006; Locato et al. 2006). In apoptosis, DNA
laddering is due to an endonuclease, called CAD [cysteinyl
Figure 1. Typical plant programmed cell death (PCD)
hallmarks. The figure reports some specific features of plant PCD
observed in tobacco cells subjected to oxidative stress conditions
inducing PCD. (a) Cytoplasm shrinkage; (b) chromatin
condensation and fragmentation; (c) DNA laddering.
Bar = 20 mm.
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Redox regulation in plant PCD 235
aspartate-specific proteinase (caspase)-activated DNase]
activated by caspase-dependent cleavage. A similar mecha-
nism for DNA fragmentation has been hypothesized in
plants (Enari et al. 1998; Li et al. 2004). Caspases represent a
family of proteases, involved in apoptosis activation, which
have cysteine in their active site and are able to induce the
cleavage in correspondence to aspartate residues (Thorn-
berry & Lazebnik 1998). In plants, no homolog of animal
caspases has been found; however, caspase-like activity has
been described in cells undergoing PCD. The vacuolar-
processing enzymes (VPEs) represent a class of plant pro-
teases, showing caspase-like activity (Hatsugai et al. 2004),
involved in virus and fungal toxin-induced hypersensitive
plant cell death (Rojo et al. 2004; Hara-Nishimura et al.
2005). The plant proteasome subunit, PBA1, has been also
indicated as a novel protease, having caspase-like activity,
involved in hypersensitive cell death (Hatsugai et al. 2009).
Proteasome activation has been shown to have an active
role in regulating PCD also in heat-shocked TBY-2 cells
(Vacca et al. 2007). However, in plants, as in animal systems,
the role of proteasome in PCD regulation is still under
debate (Kurepa & Smalle 2008; Bader & Steller 2009).
Metacaspases represent another family of cysteine pro-
teases that show a caspase-like proteolytic domain but dif-
ferent substrate specificity, since they cleave protein at the
level of arginine and lysine residues (Uren et al. 2000; Ver-
cammen et al. 2004). In plants, two types of metacaspases
have been identified (types I and II) containing a conserved
putative catalytic domain but differing in their N-terminal
domains. Type II metacaspases have been shown to be
involved in PCD occurring during embryogenesis in Picea
abies (Suarez et al. 2004) and in PCD induced by oxidative
stress in Arabidopsis (He et al. 2007). Recently, the type I
metacaspase AtMC1 has been found to be a positive regu-
lator of the hypersensitive cell death in Arabidopsis (Coll
et al. 2010).
The release of cytochrome c (cyt c) from mitochondria is
one of the biochemical events characterizing animal apop-
tosis (Balk, Leaver & McCabe 1999; Sun et al. 1999), being
required for the formation of the apoptosome, a molecular
system activating caspases (Zou et al. 1999). Although evi-
dence for the apoptosome formation is lacking in plants, the
release of cyt c has also been well documented in plant PCD
(Robson & Vanlerberghe 2002; Vacca et al. 2006; Andronis
& Roubelakis-Angelakis 2010).
In animal cells, it has been suggested that the release of
mitochondrial factors into the cytosol is regulated by the
presence of proteins of the Bcl-2 family associated with the
outer mitochondrial membrane (OMM) (Brenner et al.
2000; Cregan et al. 2002). The pro-apoptotic proteins of the
Bcl-2 family, called Bax-like, can form pores in OMM by
their oligomerization (Kroemer & Reed 2000), and the
anti-apoptotic Bcl-2 members can be considered their
antagonists. The balance between pro-apoptotic and anti-
apoptotic factors seems to be crucial for triggering apopto-
sis or mechanisms leading to cell survival (Huang 2000).
Plant factors homolog to Bcl-2 family members have not
been identified up to now. However, the expression of

236 M. C. de Pinto et al.
animal pro-apoptotic proteins in plants induces cell death
(Lacomme & Santa Cruz 1999), and transformed plants
expressing anti-apoptotic factors increase their resistance
to abiotic and biotic stresses (Mitsuhara et al. 1999). More-
over, plant homologs have been identified for other pro-
teins involved in the regulation of apoptosis, such as
defender against apoptotic death-1 (DAD-1), Bax
inhibitor-1 (BI-1) and 3 P53-induced gene (PIG; P53 is an
apoptotic protein involved in cancer) (Nakashima et al.
1993; Venot et al. 1998; Danon et al. 2000; Chae et al. 2003).
BI-I has recently been reported as a key factor, localized in
the endoplasmic reticulum (ER), controlling calcium flux
during PCD both in animal and plant models (Ihara-Ohori
et al. 2007; Kim et al. 2008). Many PCD mediators have been
reported to be localized in ER, both in plants and in
animals, suggesting that this cellular compartment could
play a crucial role in PCD (reviewed by Cacas 2010).
Recently, in plants, as in animal systems, autophagic PCD
has been suggested as an alternative form of PCD, activated
antagonistically to the apoptotic-like form (Love, Milner &
Sadanandom 2008). The term autophagy is literally related
to a self-eating process. As observed in animals, in plants,
autophagic processes are activated in starving conditions
with the aim of recycling nutrients already present in the
cell (Inoue & Moriyasu 2006). Autophagy requires the
expression of specific genes, called autophagy genes, firstly
identified in Saccharomyces cerevisiae (Tsukada & Ohsumi
1993). Many orthologs of the yeast autophagy genes have
been found in the Arabidopsis thaliana genome (Meijer
et al. 2007). Plant cells can activate autophagy not only in
order to recover energy in drastic nutrient depletion but
also as a mechanism controlling PCD. In particular, autoph-
agy seems to be required to block HR spreading since it has
been shown that autophagy defective mutants show unre-
stricted cell death after avirulent pathogen treatment (Liu
et al. 2005). In this context, autophagy could be required in
order to segregate diffusible pro-death signals coming from
the infection site. However, it can also have a pro-death role
in HR actuation. Indeed, Arabidopsis autophagy mutants,
treated with different avirulent pathogens, exhibit a
reduced expansion in HR compared to the wild type
(Hofius et al. 2009).
In plants, as in animals, the variety of genetic pathways
involved in different forms of PCD often overlaps. The
duration and the time at which different metabolic path-
ways are activated are crucial factors in determining the
activation of a specific response (de Pinto et al. 2006; Love
et al. 2008).
Reactive oxygen species and PCD
Reactive oxygen species (ROS) have been proposed as key
inducers of different types of developmental and/or envi-
ronmental PCD. However, the presence of different kinds
of ROS, different sites of ROS production as well as their
interaction with other molecules makes understanding of
the ROS signalling network, during PCD, very complex and
often poorly characterized.
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It is known that ROS are produced in plants, and other
aerobic organisms, as a result of O2 reduction during a
number of normal metabolic processes. These harmful and
highly reactive intermediates of O2 reduction, being able to
damage biological molecules, have been only considered as
unwelcome by-products of metabolism for a long time
(Möller, Jensen & Hansson 2007).
For this reason, the first indications for involvement of
ROS in PCD were based primarily on their ability to
provoke oxidative damage to cells that inevitably induce
death. The initial experimental evidence that ROS could
also act as signals in plant PCD, and not only as a mere
activator of oxidative processes, was obtained by the dem-
onstration that H2O2-induced cell death could be blocked
by inhibitors of protein synthesis (Levine et al. 1994). Since
then, several biochemical and genetic findings have under-
lined the key role of ROS as triggers of PCD. PCD con-
trolled by ROS occurs during developmental processes as
the aleurone cell death and leaf senescence, various forms
of abiotic stress, the hypersensitive response and allelo-
pathic plant–plant interactions (Bethke & Jones 2001; Bais
et al. 2003; Apel & Hirt 2004).
The chemical nature of ROS seems to be critical for
the specificity and selectivity of ROS signals during
PCD. PCD can be initiated by all types of ROS, but the roles
of different ROS remain unclear, although it has been
shown that different ROS can activate distinct signalling
pathways (Gadjev et al. 2006). In Arabidopsis plants, cell
death associated with 1O2-induced oxidative stress, previ-
ously attributed to physicochemical damage, is the result of
an active genetic programme that requires a chloroplast
protein, EXECUTER1. EXECUTER1 acts together with
EXECUTER2 to transfer the stress-related signals from
the plastid to the nucleus (Lee et al. 2007). EXECUTER2
works as a modulator controlling the activity of
EXECUTER1 subsequently to enzymatic lipid peroxida-
tion events (Przybyla et al. 2008).
Cell death observed in Arabidopsis lsd1 mutants, in the
absence of pathogen, reflects an abnormal accumulation of
superoxide and lack of responsiveness to signals derived
from it. In these mutants, superoxide is necessary and suf-
ficient to initiate lesion formation; it accumulates before the
onset of cell death and, thereafter, in cells adjacent to
spreading lsd1 lesions (Jabs, Dietrich & Dangl 1996). LSD1
codes for a zinc finger transcription factor that acts as a
negative regulator of pro-death signals (Dietrich et al. 1997;
Kaminaka et al. 2006). Interestingly, LSD1 has been shown
to interact with the LSD1-like zinc-finger N-terminal motif
of AtMC1, controlling its pro-death effects. Consistently,
the morphological lsd1 phenotypes are abolished in the
absence of AtMC1 (Coll et al. 2010).
The most studied ROS signal in PCD is H2O2 (Gechev &
Hille 2005). One well-described example of H2O2-induced
PCD in development is aleurone cell death. During seed
germination, cells of the aleurone layer metabolize their
carbohydrate reserves by means of gibberellic acid-
dependent synthesis of alpha-amylase, and this process is
rapidly followed by cell death (Bethke & Jones 2001). This

cell death is dependent on glyoxysomal production of H2O2.
Another well-studied type of PCD which might depend on
O2-/H2O2 is that accompanying the defence process
so-called HR (Heath 2000; Zurbriggen et al. 2009; Torres
2010). During HR, a biphasic burst of ROS production
occurs (Levine et al. 1994). Several enzymatic systems may
contribute to this overproduction of ROS. Cell wall-
associated peroxidases have been reported to be respon-
sible for ROS generation in the HR triggered in several
plant–pathogen interactions (Bestwick, Brown & Mansfield
1998; Martinez et al. 1998; Bindschedler et al. 2006; Choi
et al. 2007). Oxalate oxidase and amine oxidases are other
apoplastic enzymes involved in the overproduction of H2O2
under pathogen attacks or elicitor treatments (Lane 2002;
Cona et al. 2006; Yoda, Hiroi & Sano 2006). Plasma mem-
brane NADPH oxidase, generating superoxide in the apo-
plastic side and similar to NADPH oxidase present in
mammalian neutrophil cells, has been characterized in
several plant species as an essential ROS-producing system
activated during the early stages of plant–pathogen interac-
tion (Sagi & Fluhr 2001; Torres, Dangl & Jones 2002).
However, whether NADPH-dependent ROS production is
crucial for triggering PCD or if it is part of the signalling
pathways associated with plant cell protection is still under
debate (Lherminier et al. 2009).
H2O2 involvement is also reported in many types of PCD
induced by abiotic stress. Indeed, H2O2 is involved in heat
shock (HS)-induced PCD in tobacco BY-2 cells (Vacca et al.
2004; Locato et al. 2008) and in the hypoxia-induced lysig-
enous aerenchyma formation in Arabidopsis (Muhlenbock
et al. 2007).
Since H2O2 can be involved in different environmental
and developmental responses, as well as in PCD, an inter-
esting question is how this small molecule controls so many
different processes. Over the last decade, the identification
of genes responding to elevated H2O2 levels (Desikan et al.
2001; Vandenabeele et al. 2003; Gechev, Minkov & Hille
2005) and mutants deficient for H2O2 signalling pathway
(Nakagami, Kiegerl & Hirt 2004; Rentel et al. 2004) has led
to a better understanding of how the ROS-signalling
network functions.
A vast network of MAPKs is involved in relaying the
H2O2 signal in plants. The Arabidopsis MAPK kinase
kinase, MEKK1, can be activated by H2O2 in a proteasome-
dependent manner. MEKK1 knockout plants accumulate
high levels of ROS and develop local lesions reminiscent of
PCD. MEKK1 is an activator of the two highly homologous
MAPK kinases, MKK1 and MKK2, that function upstream
with regard to the MAPKs MPK4 and MPK6 (Teige et al.
2004). Since MKK1 is required for H2O2-induced activation
of MPK4, but not for MPK6, a major role of MEKK1-
MKK1/2-MPK4 cascade in ROS signalling has been pro-
posed (Nakagami et al. 2006; Pitzschke et al. 2009). MEKK1
can also interact directly with WRKY53, a transcription
factor involved in senescence-induced PCD, thus bypassing
downstream kinases (Miao et al. 2007). A role for MAPK
kinase cascades has also been shown during PCD triggered
by chloroplast-derived H2O2 in tobacco (Liu et al. 2007).
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Redox regulation in plant PCD 237
Another essential component of the H2O2 signalling
network in Arabidopsis is the serine/threonine kinase
OXI1, inducible by abiotic stress and H2O2 and required for
full activation of AtMPK3 and AtMPK6 (Rentel et al.
2004). OMTK1 is another specifically H2O2-induced kinase
identified in alfalfa, which is able to activate downstream
the MAP kinase MMK3 (Nakagami et al. 2004).
Nucleotide diphosphate kinases and protein phos-
phatases are other components of the H2O2 signalling
network (Fukamatsu, Yabe & Hasunuma 2003; Moon et al.
2003; Schweighofer, Hirt & Meskiene 2004).
Finally, the H2O2 signalling network transmits the signal
to ROS-specific transcription factors that then regulate
gene expression and lead to activation of the H2O2-
dependent cell death (Gadjev et al. 2006).
To complicate the understanding of the ROS-dependent
PCD network, there is the importance of timing and inten-
sity of oxidative stress needed for PCD induction. In
tobacco BY-2 cells, it has been demonstrated that the meta-
bolic responses activated in cells differ consistently depend-
ing both on the intensity of the oxidative stress generated
and the different timing of ROS production (de Pinto et al.
2006). The prolonged production of H2O2, for several hours,
triggers a necrotic process. On the other hand, an amount of
H2O2 comparable to that inducing cell necrosis, but given as
a single pulse, triggers PCD. Direct addition of H2O2 prob-
ably leads to a situation similar to the oxidative burst
induced by various forms of biotic or abiotic stress against
which PCD is frequently activated (Overmyer, Brosché &
Kangasjärvi 2003). Moreover, during plant cell death, a
strong interplay exists between ROS and other signalling
molecules, such as nitric oxide (NO), redox metabolites,
lipid messengers or plant hormones, that modifies the bio-
logical response to altered ROS levels and determines cell
fate (Foyer & Noctor 2009).
It is well known that treatment with NO generators
results in vanishing or increasing ROS effects (Delledonne
et al. 2001; Orozco-Cárdenas & Ryan 2002; Murgia et al.
2004), further supporting the view that signalling pathways,
based on the same chemical messengers, interplay differ-
ently depending upon the different environmental or cellu-
lar contexts. The balance between ROS and NO has also
been proposed to be a critical aspect in inducing PCD
(Delledonne et al. 1998, 2001). Recently, the crosstalk
between NO and H2O2 during plant cell death has been
consolidated by the study of the early transcriptional
responses to these reactive species (Zago et al. 2006). Part
of the NO-dependent signalling pathways passes through
post-translational protein modification (Leitner et al. 2009).
For instance, it has been shown that protein S-nitrosylation
is involved in PCD (Belenghi et al. 2007; Leitner et al. 2009).
Moreover, in Arabidopsis cells undergoing hypersensitive
cell death, the increase in peroxynitrite, due to the simulta-
neous production of NO and superoxide, is responsible for
tyrosine nitration and change in the function of several
proteins (Gaupels et al. 2011).
Despite extensive research on the source of ROS
in plant cell death, the sub-cellular location and the

238 M. C. de Pinto et al.
mechanism of ROS generation, during PCD, still remains
to be clarified.
As already pointed out, the overproduction and rapid
accumulation of ROS during HR can be caused by the
plasma membrane NADPH oxidase, or apoplastic enzymes.
Chloroplasts are involved in the regulation of stress
responses, including PCD, acting as sensors of environmen-
tal stress, and are able to increase their ROS production
(Mullineaux & Karpinski 2002). A number of studies have
shown that plant PCD, including hypersensitive cell death, is
affected by light (Genoud et al. 2002; Danon et al. 2004; Zeier
et al. 2004; Dzyubinskaya et al. 2006). It has been found that
the HR is accelerated by the loss of chloroplast function (Seo
et al. 2000) and several recent studies have linked
chloroplast-produced ROS with the HR (Mur et al. 2008).
Chloroplast-produced ROS have been shown to be also able
to transmit the spreading of wound-induced PCD through
maize tissue (Gray et al. 2002). The mitochondrion has also
been shown to be involved in ROS-induced PCD. It has been
demonstrated that cyt c is released from mitochondria fol-
lowing death stimuli such as HS, d-mannose, menadione,
harpin or ceramide treatment (reviewed in Reape &
MacCabe 2010). Release of cyt c was also found to be
associated with premature induction of PCD in the tapetum
of sunflower cytoplasmic male sterility mutants (Balk &
Leaver 2001) and death of poppy pollen tubes during self-
incompatibility (Thomas & Franklin-Tong 2004). The
release of cyt c from the mitochondria could result in disrup-
tion of electron transport leading to generation of lethal
levels of ROS (Jones 2001).Alternatively, the ROS produced
by electron transport impairment could contribute to mito-
chondrial retrograde regulation (MRR), aimed at activating
a pro-survival response (Rhoads and Subbaiah 2007 and
references therein). It has been reported that high levels of
salicylic acid (SA) may induce cell death, inhibiting mito-
chondrial electron transport via ubiquinone reduction. Elec-
tron upsurge in the electron transport chain leads to cell
death via redox stress and hyperaccumulation of ROS
(Amirsadeghi et al. 2006; Garcia-Heredia et al. 2008).
However, plants can induce mitochondrial alternative
oxidase (AOX), probably through MRR. The activity of
AOX significantly reduces electron build-up during SA
accumulation, thus reducing redox stress and ROS accumu-
lation (Norman et al. 2004). Consistently, down-regulation of
AOX, via either inhibitors or a transgenic approach, stimu-
lates PCD and leads to activation of senescence-associated
marker genes (Maxwell, Nickels & McIntosh 2002).
A role for peroxisomes and photorespiratory H2O2 has
been suggested, in lesion formation, in the lsd1 mutant of
Arabidopsis during avirulent pathogen infection (Mateo
et al. 2004). The potentially important role of the peroxi-
somes in ROS production is also highlighted in Arabidopsis
cat2 mutants, in which SA, among other responses, induces
cell death (Chaouch et al. 2010; Mhamdi et al. 2010).
Despite a plethora of results indicating the active
involvement of ROS in the signalling pathway leading to
PCD, situations in which PCD occurs independently of
ROS production have also been reported. Carrot cells with
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a reduced expression of the topoisomerase I gene activates
PCD in the absence of an overproduction of H2O2 (Locato
et al. 2006). Dihydrosphingosine, a sphingolipid accumu-
lated after infection of necrotrophic fungi and ophiobolin
A, a fungal sesquiterpenoid phytotoxin, also induces H2O2-
independent PCD (Lachaud et al. 2011; De Gara & Locato,
unpublished results), thus making the ROS-dependent
signalling pathways leading to PCD more intriguing.
Role of antioxidants in ROS-induced PCD
The level of ROS in a cell depends on a balance between
ROS-producing and ROS-scavenging systems. Balancing
ROS levels is essential to ensure accurate execution of sig-
nalling functions and to prevent toxicity. Therefore, plants
have produced an elaborate antioxidant system, consist-
ing of enzymes and non-enzymatic antioxidants, which,
together with the ROS producing sources, maintain ROS
homeostasis in all cell compartments and adjust ROS levels
according to the cell need at a particular time.Thus, reduced
efficiency of ROS scavenging, by metabolites and enzymes,
would be expected to play a major role in onset of PCD.
In the H2O2- and HS-induced PCD, ascorbate (ASC)
level, more than its redox state, is altered as a specific signal
in TBY-2 cells undergoing PCD (de Pinto et al. 2006; Locato
et al. 2008). Consistently, A. thaliana mutants, with 10–25%
of wild-type ASC content but a normal ASC redox state,
spontaneously activate processes that typically occur during
HR, such as localized cell death and the expression of
pathogenesis-related proteins (Pavet et al. 2005).
The decrease in ASC observed in TBY-2 cells undergoing
HS-induced PCD could be due, at least in part, to an impair-
ment of L-galactone-g-lactone dehydrogenase (GLDH),
the last enzyme of ASC biosynthesis (Valenti et al. 2007). It
has been suggested that GLDH is an integral part of plant
mitochondrial complex I, the redox state of which affects
GLDH catalysis (Millar et al. 2003). GLDH inhibition could
possibly be a consequence of complex I inhibition. What-
ever the cause of GLDH inhibition, it results in a decrease
in the amount of ASC available to counteract ROS. It is
worthwhile to point out that in spite of the fact that the ASC
pool of the whole cells is only moderately decreased after
2 h of PCD induction, the GLDH activity is much strongly
decreased (Vacca et al. 2004). This probably results in
depletion in the ASC pool and a decrease in the ROS-
scavenging system, faster in mitochondria than in other cell
compartments.
An impairment of enzymes involved in H2O2 removal has
been reported for various kinds of PCD. During monocot
seed germination, in the aleurone layer, the glyoxysomal
antioxidant enzymes, catalase (CAT), ascorbate peroxidase
(APX) and superoxide dismutase, are down-regulated by
gibberellic acid to ensure sufficient accumulation of H2O2
prior to the onset of cell death (Fath, Bethke & Jones 2001).
Transgenic tobacco plants, with reduced CAT activity, accu-
mulate high levels of H2O2 under photorespiratory condi-
tions. This perturbation in H2O2 homeostasis induces cell
death in clusters of palisade parenchyma cells (Dat et al.

Figure 2. APX regulation during oxidative conditions leading
to programmed cell death (PCD). The figure summarizes the
results obtained by the authors. Tobacco cells subjected to direct
oxidative stress or heat shock-derived oxidative conditions can
activate PCD through a signalling pathway involving the
regulation of cytosolic APX at different levels (changes in
kinetics characteristics, protein nitrosylation/oxidation, protein
turn-over and alteration in gene expression). CAT, catalase; NO,
nitric oxide; ROS, reactive oxygen species.
2003). Arabidopsis plants overexpressing or underexpress-
ing one thylakoidal APX gene present increased or
decreased sensitivity to NO-induced cell death (Tarantino
et al. 2005). A key role for cytosolic APX down-regulation
in generating the conditions needed for HR has been
reported in the interaction between tobacco and tobacco
mosaic virus (TMV). The APX was suppressed at the trans-
lational level during TMV-induced PCD; moreover,
transgenic antisense plants with reduced APX were hyper-
responsive to TMV attacks (Mittler, Feng & Cohen 1998;
Mittler et al. 1999).
Regulation of the cytosolic APX, in H2O2- and HS-
induced PCD, in TBY-2 cells has been studied more in
detail, and this enzyme has been defined as a key regulator
of PCD (Vacca et al. 2004; de Pinto et al. 2006). Indeed, the
enzyme is subjected to various regulatory mechanisms,
which are activated at different times (Fig. 2). Immediate
regulation occurs by changes in the kinetic capacity of the
enzyme (Vacca et al. 2004; de Pinto et al. 2006). The change
from sigmoidal to hyperbolic kinetics could be a conse-
quence of NO generation observed during these PCDs. It is
well known that the formation of S-nitrosothiol groups is
part of the signalling pathways triggered by NO in animals
and plants (Hofmann, Ammendola & Schlossmann 2000;
Belenghi et al. 2007; Leitner et al. 2009). The fact that APX
has a cysteine close to catalytic site makes it a putative NO
target (Lad, Mewiers & Lloyd Raven 2002). During H2O2-
induced PCD, the decrease in the glutathione (GSH) pool
observed is due to the production of S-nitrosoglutathione
which is a powerful nitrosylating agent (Ji et al. 1999) that
might contribute to promote APX nitrosylation responsible
for the change of the kinetics characteristics of the enzyme
© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 234–244
13653040, 2012, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.2011.02387.x by INASP/HINARI - PAKISTAN, Wiley Online Library on [09/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Redox regulation in plant PCD 239
(de Pinto, Locato & De Gara, unpublished results). It has
consistently been reported that NO production negatively
affects CAT and APX activities in Cd-induced PCD in Ara-
bidopsis suspension cultures (De Michele et al. 2009). A
second level of cytosolic APX regulation during PCD con-
cerns protein turnover which probably involves ubiquitin–
proteasome-dependent protein degradation (Vacca et al.
2007). Last but not least, the cell environment determined
by the simultaneous presence of NO and H2O2 during PCD
also appears to affect APX expression, which is significantly
reduced a few hours after PCD activation (Vacca et al. 2004;
de Pinto et al. 2006).
Data on the involvement of different organellar isoen-
zymes of the ASC-GSH cycle during HS-induced PCD have
also been reported (Locato, de Pinto & De Gara 2009). A
decrease in the different APX isoenzymes was observed,
indicating that APX plays a key role in promoting the oxi-
dative burst needed for PCD in all the cell compartments.
The activities of ASC-GSH recycling enzymes increase con-
siderably in the cytosolic fraction probably to compensate
for the depletion in the redox metabolites ASC and GSH. In
the mitochondria and plastids,the activities of theASC-GSH
recycling enzymes decrease, with the exception of the mito-
chondrial dehydroascorbate reductase (DHAR). These
results indicate that the various cell compartments are spe-
cifically affected even if the same stress is exerted on the
entire cell. Moreover, the cytosol seems to be particularly
sensitive, playing a crucial role in controlling redox homeo-
stasis. It is worth noting the behaviour of mitochondrial
monodehydroascorbate reductase (MDHAR), the only
ASC recycling isoenzyme, whose activity was no longer
detectable in the cells undergoing PCD. It has been reported
that MDHAR is a homolog of the apoptosis-inducing factor
(AIF), a flavoprotein with NADH oxidase activity, that can
translocate from the mitochondrial intermembrane space to
the nucleus upon induction of apoptosis, where it induces
chromatin condensation and cleavage of DNA in large frag-
ments (Susin et al. 1999). The release of AIF, with MDHAR
activity, might explain the clearly evident drop in the activity
of the enzyme, observed in the mitochondria of cells under-
going PCD. In 55 °C heat-shocked mitochondria devoid of
MDHAR and with reduced ASC biosynthetic capability, the
role of DHAR seems to be particularly relevant. In fact, this
enzyme might considerably improve the use of the ASC still
available in a cell organelle that is strongly affected by
oxidation dependent on the ROS overproduced in situ.
Therefore, the increase in DHAR activity could be a kind of
feedback regulation mechanism which would aim at improv-
ing ASC regeneration from DHA when ASC depletion
occurs at the production site.
Other redox pathways are involved in PCD actuation,
probably working in synergy with ASC. For instance, the
GSH level and redox state in mitochondria and cytosol
modify the cyt c-dependent activation of cell death (Ghi-
belli et al. 1999; Hancock, Desikan & Neill 2001). GSH also
plays a pivotal role for the post-translational modifications
of cysteine residues (reversible oxidation, nitrosylation, glu-
tathionylation) that, in turn, regulate protein activity or

240 M. C. de Pinto et al.
turnover. A recent meta-analysis of data representative of
13 plant and fungal orders shows that glutathione redox
state is pivotal for cell survival: when GSSG:2GSH ratio
overcomes a threshold value, the PCD transduction signal-
ling is activated (Kranner et al. 2006). However, the rel-
evance of the perturbation in glutathione level and redox
state, for cell death, is still under debate (Foyer & Noctor
2011). GSH and peroxiredoxin are also involved in protect-
ing DNA from ROS even if it has recently been suggested
that cytosolic APX1 and peroxisomal CAT2 play a major
role in protecting DNA as well as in blocking PCD induc-
tion under photo-oxidative stress in Arabidopsis plants
(Vanderauwera et al. 2011).
CONCLUSIONS
It is noteworthy that many of the molecular players
involved in PCD have pleiotropic functions with important
roles in the control of vital processes, and this is particularly
true for all the metabolites and enzymes involved in the
redox network. The balance and the interaction between
ROS and antioxidant systems are pivotal for generating a
plethora of signals triggering different pathways that decide
cell fate. The activation or the silencing of these different
metabolic routes is crucial for maintaining organ/organism
functionality that can also require the death of a certain
number of cells. Almost all cellular compartments contrib-
ute to modulate endogenous and exogenous signals deter-
mining a complex crosstalk that finally establishes the most
suitable response. The increasing evidence of the central
role of redox regulation in such crosstalk further underlines
the importance of redox systems for plant development and
fitness.
ACKNOWLEDGMENT
The work of the authors was supported by the Italian
Ministry of University and Research (PRIN n.
20084XTFBC_001).
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Received 31 April 2011; received in revised form 13 June 2011;
accepted for publication 14 June 2011