Journal of Experimental Botany Advance Access published March 7, 2015 Journal of Experimental Botany doi:10.1093/jxb/erv083

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, Indiana 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

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Page 2 of 8 | 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  |  Page 3 of 8 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

Page 4 of 8 | 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  |  Page 5 of 8

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).

Page 6 of 8 | 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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