Journal of Experimental Botany, Vol. 66, No. 10 pp. 2869–2876, 2015
doi:10.1093/jxb/erv083 Advance Access publication 7 March 2015
REVIEW PAPER
The role of reactive oxygen species and nitric oxide in
programmed cell death associated with self-incompatibility
Irene Serrano*, María C. Romero-Puertas, Luisa M. Sandalio and Adela Olmedilla
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1,
E-18008 Granada, Spain
* Present address and to whom correspondence should be sent: Department of Biology, Indiana University, Bloomington, IN 47405,
USA. E-mail: [email protected]
Received 5 December 2014; Revised 20 January 2015; Accepted 9 February 2015
Abstract
Successful sexual reproduction often relies on the ability of plants to recognize self- or genetically-related pollen and
prevent pollen tube growth soon after germination in order to avoid self-fertilization. Angiosperms have developed
different reproductive barriers, one of the most extended being self-incompatibility (SI). With SI, pistils are able to
reject self or genetically-related pollen thus promoting genetic variability. There are basically two distinct systems
of SI: gametophytic (GSI) and sporophytic (SSI) based on their different molecular and genetic control mechanisms.
In both types of SI, programmed cell death (PCD) has been found to play an important role in the rejection of selfincompatible pollen. Although reactive oxygen species (ROS) were initially recognized as toxic metabolic products,
in recent years, a new role for ROS has become apparent: the control and regulation of biological processes such as
growth, development, response to biotic and abiotic environmental stimuli, and PCD. Together with ROS, nitric oxide
(NO) has become recognized as a key regulator of PCD. PCD is an important mechanism for the controlled elimination of targeted cells in both animals and plants. The major focus of this review is to discuss how ROS and NO control
male-female cross-talk during fertilization in order to trigger PCD in self-incompatible pollen, providing a highly effective way to prevent self-fertilization.
Key words: Ca2+, nitric oxide (NO), Olea europaea L., Papaver rhoeas L., peroxynitrite, pollen, programmed cell death (PCD),
Pyrus pyrifolia L., reactive oxygen species (ROS), self-incompatibility (SI).
Introduction
Plants, as sessile organisms, have developed various mechanisms to optimize mating. The majority of angiosperms are
hermaphroditic and, although the possibility of self-fertilization can be beneficial for ensuring reproduction when mates
or pollinators are not readily available, most plants have
developed mechanisms that promote out-crossing. One of the
most widespread intraspecific barriers for avoiding self-fertilization in angiosperms is self-incompatibility (SI) (Clarke
and Gleeson, 1981; de Nettancourt, 1997; Igic et al., 2004).
This mechanism permits the pistil to discriminate between
self-pollen and non-self-pollen and to mediate the rejection
of self-pollen. It has been found that self-incompatibility
systems arose quite late in evolution, and that is why closely
related families do not share homologous systems (Matton
et al., 1994; Igic and Kohn, 2001). Thus, studies of SI cannot be limited to model plants. It is necessary to extend these
studies to other species of economic significance in order to
increase their production or to facilitate their hybrid breeding. In spite of these differences, several studies carried out
in different species belonging to the two main systems of
SI (gametophytic and sporophytic), have shown that programmed cell death (PCD) is triggered in self-incompatible
pollen after pollen–pistil interactions. PCD hallmarks have
been found in pollination assays carried out in vitro as well
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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2870 | Serrano et al.
as in vivo in distantly related species, such as poppy (Papaver
rhoeas L.), pear (Pyrus pyrifolia L.), and olive (Olea europaea
L.) (Bosch and Franklin-Tong, 2008; Serrano et al., 2010;
Wang et al., 2010). More recently, alterations in the integrity
of F-actin cytoskeleton, which could trigger PCD, have been
described in self-incompatible pollen in Nicotiana alata L.
(Roldán et al. 2012, 2015).
Reactive oxygen species (ROS) and nitric oxide (NO) have
been described as key molecules for the triggering and development of different types of PCD induced in plants during
their normal development or as a response to different stress
treatments (Neill et al., 2003; de Pinto et al., 2012; Wang
et al., 2013). In this review, an attempt has been made to present the current knowledge on ROS and NO involvement in
PCD triggered after pollination in self-incompatible pollen.
ROS and NO as signalling molecules
in plants
Reactive oxygen species (ROS) are chemically reactive molecules derived from oxygen as a consequence of cellular
metabolism in aerobic organisms (Halliwell, 2006; Halliwell
and Gutteridge, 2007). Since an excessive accumulation of
these ROS has extremely harmful effects (Gechev et al., 2006;
Sharma et al., 2012), plants have evolved an effective antioxidant system, including antioxidant molecules, antioxidant
enzymes and detoxifying enzymes, which protects them from
oxidative damage, thus balancing production and removal of
the ROS (Gechev et al., 2006; Wrzaczek et al., 2013). The most
common reactive oxygen species are the free radicals superox.
ide anion ( O.−
2 ) and hydroxyl radical ( OH) and the non-radical molecules hydrogen peroxide (H2O2) and singlet oxygen
(1O2) (Apel and Hirt, 2004; Gechev et al., 2006; Sharma et al.,
2012). Chloroplasts, mitochondria, and peroxisomes are the
major sources of ROS production in plant cells (Suzuki et al.,
2012; Sharma et al., 2012; Sandalio et al., 2013).
Although early research involving ROS was frequently
associated with cytotoxicity, over the last decade, the role of
ROS has been completed and it has become evident that they
can also function as signalling molecules in a wide variety
of cellular processes (Foyer and Noctor, 2005; Mittler et al.,
2011; Wrzaczek et al., 2013; Baxter et al., 2014). For the use
of ROS as damaging or signalling molecules a tight balance
between ROS production and scavenging is necessary (de
Pinto et al., 2012; Sharma et al., 2012; Baxter et al., 2014).
This delicate balance enables rapid and dynamic changes
in ROS levels, which triggers different signalling networks
depending on different factors such as: the chemical identity of ROS, local concentration of these radicals, intensity
and duration of the signal, the site of ROS production, the
developmental stage of the plant, and interaction with other
signalling molecules such as lipid derivatives, hormones, and
nitric oxide (Gechev et al., 2006; Vellosillo et al., 2010; Mittler
et al., 2011; Chaudhuri et al., 2013; Baxter et al., 2014; Mor
et al., 2014). Recent examples of such specificity are shown by
Sewelam et al. (2014), where they demonstrate that H2O2 produced in either chloroplasts or peroxisomes is able to trigger
different cellular responses; and by Rosenwasser et al. (2011)
where they discovered that early responses to long periods of
dark are centred in the mitochondria and peroxisome, and
that these organelles are the source of senescence signalling
induced by darkness.
The first report of nitric oxide (NO) generation within a
biological system was carried out in plants (Klepper, 1979),
but it was not until 1996 that NO was reported to be involved
in plant immunity in potato (Noritake et al., 1996). Shortly
after, it was shown that NO is a key molecule in the defence
response in Arabidopsis and tobacco plants (Delledonne
et al., 1998; Durner et al., 1998). Although NO can have, as
do ROS, a damaging effect depending on the rate/place of
production (Beligni and Lamattina, 1999), a large body of
evidence has accumulated supporting its role as a signalling
molecule in physiological processes such as plant growth and
development, as well as in response to numerous biotic and
abiotic stresses (Beligni and Lamattina, 2000; Besson-Bard
et al., 2008; Fernández-Marcos et al., 2012; Chen et al., 2014;
León et al., 201; Yu et al., 2014). NO has also been shown to
be involved in the establishment of symbiotic interactions and
in root nodule senescence (del Giudice et al., 2011; Cam et al.,
2012). It is widely accepted that NO and reactive nitrogen
species (RNS) regulate different processes by inducing gene
transcription, activating secondary messengers or directly
regulating proteins (Palmieri et al., 2008; Besson-Bard et al.,
2008; Gaupels et al., 2011; Martinez-Ruiz et al., 2011). The
identification of direct targets of NO through S-nitrosylation
or nitration under physiological or stress conditions, and the
characterization of some of them, has led to great progress
in the knowledge of NO-dependent signalling mechanisms
(Vandelle and Delledonne, 2011; Astier et al., 2012; Yu et al.,
2012; Kovacs and Lindermayr, 2013; Romero-Puertas et al.,
2013).
The interplay between RNS and ROS and their balance
has been reported to be an important factor in the fate of
cells in both physiological and stress conditions (Delledonne
et al., 2001; Neill et al., 2008; Rodriguez-Serrano et al., 2009).
In fact, ROS producing antioxidant enzymes are targets of
S-nitrosylation, suggesting a fine-tune regulation of NO/ROS
balance (de Pinto et al., 2013; Romero-Puertas et al., 2013).
PCD in plants: ROS and RNS function.
PCD is an active and genetically controlled form of cell death.
PCD is a fundamental cellular process that occurs throughout
plant life, being essential not only for normal development,
but also in response to biotic and abiotic stresses (Pennell and
Lamb, 1997; van Doorn, 2005; Gechev et al., 2006; Bozhkov
and Lam, 2011).
Different types of PCD have been found in plants, and
several attempts have been made to categorize them via different studies (van Doorn and Woltering, 2005; Reape et al.,
2008; van Doorn, 2011; van Doorn et al., 2011). Although
the molecular understanding of plant cell death regulation
is still largely unknown, morphological criteria, such as
altered nuclear morphology, vacuolar, mitochondrial, and
ROS and NO in programmed cell death associated with self-incompatibility | 2871
endoplasmic reticulum swelling, protoplast shrinkage, and
cytoskeleton reorganization have been used to classify plant
cell death scenarios. Other non-morphological hallmarks
used to define types of plant PCD are DNA fragmentation,
caspase-like activity, and ROS and RNS accumulation. In an
effort to simplify plant PCD, two types of PCD have been
described: autolytic and non-autolytic PCD (van Doorn et al.,
2011). Autolytic PCD is associated with a gradual decrease in
the volume of the cytoplasm and a concomitant increase in
the volume of the lytic vacuole; there is a release of hydrolases from the vacuole after vacuole collapse, which results
in rapid clearance of the cytoplasm; this type of cell death is
always associated with the presence of autophagic-like structures in the cytoplasm. Non-autolytic PCD could involve
vacuole collapse, but it is not accompanied by rapid clearance
of the cytoplasm and thus does not resemble autophagy (van
Doorn, 2011). There are some examples of plant PCD that
cannot be described by one of these two major classes, such
as the case of PCD in pollen as a result of self-incompatibility which exhibits some characteristics of autolytic PCD
such as vacuole enlargement, but also includes characteristics
of non-autolytic PCD, such as swelling of the mitochondria
(van Doorn and Woltering, 2005; Bosch and Franklin-Tong,
2008; van Doorn, 2011).
ROS and NO are important players that are required for
PCD in plants (De Pinto et al., 2012). The PCD associated with
the hypersensitive response (HR) is one of the best characterized and the role of H2O2 and NO as key signalling molecules
inducing HR is well-established (Levine et al., 1994; Lamb
and Dixon, 1997; Grant and Loake, 2000). The ratio of NO
to H2O2 determines when cell death is activated (Delledonne
et al., 2001). Ozone is also used as a model of cell-death regulation where the ROS produced from the degradation of O3
in the apoplast appears to enhance the HR programme, and
where ROS are involved in both the initiation and propagation of cell death (Overmyer et al., 2003). During this process,
an accumulation of NO has been observed before ethylene
(ET), jasmonic acid (JA), and salicylic acid (SA) accumulation suggesting a role also for this signalling molecule in the
O3-induced PCD (Ahlfors et al., 2009). PCD also occurs as a
consequence of several other abiotic forms of stress (de Pinto
et al., 2012) and the involvement of ROS and RNS has been
described in some of them. Thus, in cadmium-induced PCD,
NO appears to control antioxidant metabolism in suspension
cell cultures (De Michele et al., 2009) and promotes MPK6mediated caspase-3-like activation in Arabidopsis (Ye et al.,
2013). In addition, cytosolic ascorbate peroxidase (cAPX), a
key enzyme regulating H2O2 levels in plants, was found to be
S-nitrosylated at the onset of heat stress and H2O2 induced
PCDs (de Pinto et al., 2013). H2O2 has also been involved in
developmental PCD in barley aleurone layers and it has been
postulated that NO may regulate this process by modulating
the antioxidant capacity of the cells (Bethke and Jones, 2001;
Beligni et al., 2002).
The role of peroxynitrite (ONOO–) in plant PCD remains
controversial. During HR, ONOO–, formed by reaction
between NO and O.−
2 , has been detected with the concomitant burst of NO and ROS produced in response to avirulent
pathogens (Vandelle and Delledonne, 2011). Although,
in animals, ONOO– induces cell death and most of the
NO-dependent cytotoxicity is attributed to this molecule
(Pacher et al., 2007), in plants, the addition of ONOO– does
not induce cell death (Delledonne et al., 2001). This may
be due to the existence of plant detoxifying systems that
rapidly remove this molecule in physiological conditions
(Sakamoto et al., 2003; Romero-Puertas et al., 2007). The
addition of urate, a ONOO– scavenger, significantly attenuates cell death during the Arabidopsis HR in response to an
avirulent pathogen (Alamillo and García-Olmedo, 2001) but
does not influence cryptogein-dependent cell death, which is
partly mediated by NO (Lamotte et al., 2004). Our group has
recently reported that an ONOO–-dependent signalling pathway mediates PCD in self-incompatible pollen and in stigmatic papillar cells (Serrano et al., 2012a, b). In these studies
it was shown that the addition of either NO or O.−
2 scavengers prevented cell death in self-incompatible pollen, and in
papillar cells after pollen arrival.
SI as a mechanism to prevent inbreeding
The success of the angiosperms as the most prosperous plant
group is due to a series of evolutionary adaptations that
favour cross-pollination and thus genetically diverse populations. Some of these barriers are physical, such as having
male and female reproductive structures on separate plants,
or temporal, in which male and female reproductive organs
from the same plant mature at different times. When both
male and female reproductive organs are in the same flower,
self-incompatibility (SI) forms partial or complete barriers
during self or related pollen tube growth in the pistil, thus
preventing self-fertilization (Clarke and Gleeson, 1981; Igic
et al., 2004).
Although all SI given definitions emphasize its role to
prevent self-fertilization, the enclosure of post-fertilization
mechanisms, such as SI, has been subject to scientific debate.
Nowadays, the most widely accepted definition for SI is ‘the
inability of fertile hermaphrodite seed plants to produce
zygotes after self-fertilization’ (de Nettancourt, 1997), which
comprised only pre-fertilization barriers.
The best-characterized SI systems are controlled by a single
highly polymorphic locus, the S-locus (de Nettancourt, 2001;
Hiscock and McInnis, 2003; McClure and Franklin-Tong, 2006;
McClure et al., 2011; Eaves et al., 2014) and they can be divided
into two categories: sporophytic self-incompatibility (SSI) and
gametophytic self-incompatibility (GSI). SSI is limited in its
distribution; it has been found in the Brassicaceae, Asteraceae,
and Convolvulaceae, but has only been studied in detail in the
Brassicaceae. The SSI mechanism of pollen rejection involves
the interaction between the stigma-expressed S-locus Receptor
Kinase (SRK), which encodes a serine/threonine receptor
kinase located at the plasma membrane of the stigma and its
pollen-coat localized ligand, the S-locus Cysteine-Rich protein
(SCR), which encodes a cysteine-rich protein (Schopfer et al.,
1999; Takayama et al., 2001; Leducq et al., 2014). Pollen will
not germinate if any alleles of the pollen parent match either
2872 | Serrano et al.
S alleles of the pistil parent. Although this is one of the few
examples of protein/peptide–ligand/receptor interacting pairs
identified in flowering plants, there are not many insights
about the mechanisms that lead to self-pollen inhibition in the
Brassicaeae (Dresselhaus and Franklin-Tong, 2013).
Better characterized mechanistically are the two GSI systems known to date. One has so far been found only in the
Papaveraceae; in this SI system, the female determinant is
a protein (PrsS) secreted to the pistil surface (Foote et al.,
1994) and the male determinant is a transmembrane protein
(PrpS) located in pollen (Wheeler et al., 2009). In this system, pollen recognition as self (genetically identical or selfincompatible), after PrsS and PrpS interactions, lead to an
increase in intracellular Ca2+, which induces a multi-layered
SI signalling cascade that culminates in PCD of incompatible
pollen (Eaves et al., 2014; Wilkins et al., 2014) (Fig. 1). The
other GSI is very widespread among angiosperms and has
been extensively studied in the Solanaceae, Plantaginaceae,
and Rosaceae (Kear and McClure, 2012). This GSI is regulated by a set of tightly linked genes at the S-locus: the
S-RNase gene, which regulates pistil specificity (Lee et al.,
1994; Murfett et al., 1994), and multiple S-locus F-box (SLF
or SFB) that collectively regulate pollen specificity (Kubo
et al., 2010) (Fig. 1). In this system of SI, the rejection of selfpollen occurs during the growth of pollen tubes in the style
and it is controlled by extracellular ribonucleases (S-RNases),
which penetrate the pollen tube and degrade the RNA from
self-pollen (Gray et al., 1991; Luu et al., 2000).
Although the SI determinants have not yet been characterized in the olive, it has been suggested that it belongs to
the group of GSI species. It shows features characteristic of
GSI species: it has bicellular pollen, wet stigma, and a solid
style and, furthermore, in spite of a large number of pollen
grains germinating in the stigma, it is rare to detect more than
one in the style. In addition, the authors’ group have detected
RNase activity in pollen tubes growing in freely pollinated
pistils and in in vitro germinated pollen in the presence of
self-incompatible pistils. These findings suggest that RNases
may well be involved in intraspecific pollen rejection in olive
flowers (Serrano and Olmedilla, 2012) (Fig. 1).
Fig. 1. Simplified representation of the most relevant molecules found to be involved in PCD induced after SI. (A) In Papaver rhoeas L. the specific
interaction between PrpS and PrsS protein triggers an increase in cytosolic Ca2+ leading to an increase in ROS/NO, which targets downstream signalling
leading to PCD. (B) In Pyrus pyrifolia L., increased levels of apoplast CaM trigger an increase in cytosolic Ca2+ leading to an increase in ROS. S-RNAses
disrupt tip-localized ROS, which result in downstream signalling leading to PCD. (C) In Olea europaea L., although the SI determinants have not yet
been characterized, an increase in NO and O.−
2 is detected in self-incompatible pollen, where NADPH oxidase and NOS like activities, which are
Ca2+dependent, have been detected. These molecules react to form ONOO–, increasing protein nitration, which may cause a destabilization of actin
filaments and thus PCD. Green lanes indicate positive regulation, red lanes negative regulation. Dashed lines indicate non-proven hypothesis.
ROS and NO in programmed cell death associated with self-incompatibility | 2873
ROS and NO orchestrate SI response in
incompatible pollen
Pollen tube germination, growth, and guidance throughout
pistil tissues is a tightly regulated process that comprises a
continuous exchange of signals, both physical and chemical
(Cheung et al., 1995; Wu et al., 1995; McInnis et al., 2006a;
Chae and Lord, 2011). First, the pollen grain has to alight on
a receptive stigma and be recognized by the stigma as a pollen
grain and not as an inappropriate invasion. It has been shown
that stigmas from different species accumulated high levels of
H2O2 when they are receptive and that these levels decrease
on stigmas supporting pollen development (McInnis et al.,
2006a, b; Hiscock et al., 2007; Zafra et al., 2010; Serrano and
Olmedilla, 2012; Serrano et al., 2012a). Different functions
have been speculated for the stigma H2O2: loosening cell-wall
components in order to allow penetration of the pollen tubes,
defence against pathogens, and pollen–pistil recognition
(McInnis et al., 2006a; Serrano and Olmedilla, 2012; Serrano
et al., 2012a) and it has been suggested that NO produced
by the pollen is the signal triggering the H2O2 decrease on
the stigma (Serrano et al., 2012a). Once the pollen grain has
germinated, NO, produced by pollen, plays a crucial role in
pollen tube navigation across the pistil tissues (Prado et al.,
2004).
To support pollen germination and fertilization, the pistil has to recognize the pollen as non self (self-compatible)
(Heslop-Harrison, 1978). During recent years, a large body
of evidence has supported a role for ROS and NO in the SI
response which ultimately triggers PCD in self-compatible
pollen (Bosch et al., 2010; Wilkins et al., 2011; Serrano et al.,
2012a, b; Jiang et al., 2014). The first data linking ROS with
the signalling events occurring during the SI response were
described in Pyrus pyrifolia L., a species with an S-RNase
based SI (Wang et al., 2010) (Fig. 1). S-RNases are able specifically to disrupt tip-localized ROS of in vitro germinated
“self ” (self-incompatible) pollen via mitochondrial alteration and a decrease in mitochondrial and cytosolic NADPH
oxidases. As a consequence of tip-localized ROS disruption, there is an alteration in intracellular Ca2+ current, a
depolymerization of the actin cytoskeleton, and nuclear
DNA degradation, hallmarks of PCD (Obara et al., 2001;
Thomas et al., 2006). A recent article by Jiang et al. (2014)
shows the interplay between Ca2+, ROS, actin filaments, and
calmodulin (CaM) in regulating pollen tube growth in pear.
This work shows that in SI pollen tubes, Ca2+ current, ROS
accumulation, and actin filament depolymerization are CaM
dependent.
ROS and NO have also been involved in the Papaver
rhoeas L. SI response (Wilkins et al., 2011) (Fig. 1). In this
work, the authors demonstrate that Papaver pollen germinated in vitro rapidly and transiently accumulates ROS and
NO, to a different extent depending on the nature of the
pollen grain (self-compatible or self-incompatible). There is
an increase in cytosolic ROS shortly after the induction of
the SI-response and, later, a transient increase in NO. The
combined pre-treatment of the NADPH oxidase inhibitor
DPI (diphenyleneiodonium) and the NO scavenger cPTIO
[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl3-oxide], drastically decreased caspase-3-like/DEVDase activity and the formation of punctate actin foci, which are key
features of the Papaver SI-PCD response (Geitmann et al.,
2000; Bosch and Franklin-Tong, 2007). The effect of ROS
and NO on the actin cytoskeleton has recently been shown to
be via post-transcriptional modification of actin by carbonylation and S-nitrosylation that interfere with actin polymerization, giving rise to severe disturbances in actin cytoskeleton
structure and function (Rodríguez-Serrano et al., 2014).
In the authors’ laboratory, the role of ROS and RNS in
the olive (Olea europaea L.) pollen–pistil interaction has
been investigated in vitro and in vivo by using free and controlled pollination. It was found that there is an exchange of
signals between the stigma and the pollen, which appears to
regulate ROS and RNS production in both tissues (Serrano
et al., 2012a) (Fig. 1). While H2O2 was found in stigma papillae from pistils before pollination, it is reduced after pollen
arrival. By contrast, an increase in O.−
2 and NO after pollination was observed with a concomitant increase in ONOO–.
It appears that both NADPH oxidase and peroxidase activities may be involved in O.−
2 production and that NOS-like
activity is involved in NO production (Serrano et al., 2012a).
Treatment with a scavenger of ONOO–completely eliminated
cell death in papillar cells and reduced the number of pollen
grains undergoing PCD. ONOO–-dependent nitration was
also found both in papillar cells of the stigma and in pollen
undergoing cell death, suggesting that a dependent nitration
and PCD signalling takes place during incompatible pollination in the olive.
Conclusions and remarks
The involvement of ROS and NO has been described in three
different SI responses: the well-characterized Papaver GSI,
the S-RNase based Rosaceae GSI, and the SI response in the
olive, which has not yet been characterized at the molecular level (Fig. 1). The connection between these different SI
responses via ROS and NO, emphasizes the importance of
these molecules in SI processes and underlines that, although
the determinants for these SI responses have diverged, the
executers of the response seem to be conserved in distant
species, indicating a potential link among the different SI
systems.
The ROS and RNS signalling mechanisms have been constructed with transcriptomic analysis (Besson-Bard et al.,
2008; Vandenbroucke et al., 2008). Both types of molecules
have also been shown to mediate different hormone-regulated
processes in plants. In addition, cross-talk between ROS,
RNS, and hormones has been described in response to environmental cues, involving second messengers such as kinases
or Ca2+ (Rodríguez-Serrano et al., 2009; Simontacchi et al.,
2013), but this cross-talk has scarcely been investigated in
SI. A further line of study should focus on direct ROS and
NO-dependent protein regulation during SI responses, since
an increase in nitration in self-incompatible pollen has been
reported during SI in the olive tree (Serrano et al., 2012a).
2874 | Serrano et al.
The identification of a number of plant proteins that are
direct targets of NO and ROS during SI and the role of
post-translational modifications of proteins by nitration,
S-nitrosylation or carbonylation would be an important clue
to characterize, both functionally and biochemically, the signalling network underlying this process.
Acknowledgements
The authors acknowledge Ms Angela Tate for revising our English text. This
work was supported by ERDF-co-financed projects from the Spanish MEC
(BFU2006- 09876/BFI and BIO2008-04067).
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