Keywords

1 Introduction

Programmed cell death (PCD) occurs in various forms throughout the plant life cycle, probably with both common and specific aspects. PCD is a genetically controlled biological process which activates an intrinsic suicide program of cells. It not only controls the degradation of intracellular components, but facilitates removal of unwanted, incorrect, or damaged cells from multicellular organisms. It plays an important role in defense response, development, and environmental stress. Leaf senescence, hypersensitive response (HR), lysigenous aerenchyma formation, and aleurone degradation are all the forms of PCD in pants.

Generally, the characters of plant PCD are similar to animal apoptosis, such as cytoplasm shrinkage, chromatin condensation, membrane blebbing, DNA fragmentation, and selective cleavage of proteins. During HR development, plant also can form apoptotic bodies. Although individual processes differ in the triggering factors such as vacuole collapse, releasing sequestered hydrolases, may be the universal trigger of plant PCD. It is indicated that the molecular machinery underlying PCD is well conserved in eukaryotic organisms. The executive phases and typical hallmarks of PCD differ under different occasions. Cleavage of genomic DNA during apoptotic PCD is divided into two subsequent steps; an early cleavage into high molecular weight fragments, whose sizes coincidence with chromatin loop domains, and later an intense fragmentation, usually forming oligonucleosomal fragments (Brotner et al. 1995), that can be detected by DNA electrophoresis in the whole tissue or cell population, also visualized by TUNEL reaction (TdT-mediated dUTP nick-end labeling) in individual cells (Gavrieli et al. 1992; Zhan et al. 2013).

Nitric oxide (NO) a simple diatomic, diffusible, gaseous free radical, involved in many physiological processes such as PCD, seed germination, lateral root initiation, flowering, stomatal closure, and responses to stress in plants. Moreover, as an important signaling molecule, NO has been implicated in environmental-induced plant PCD, but its signaling network is still unknown. Whether NO promotes or suppresses PCD is dependent on sources and concentration of NO in different plant species.

2 Evolution of NO and Dual Function During Plant Programmed Cell Death

Owing to the essential function of NO in plant signaling network, its endogenous source is very important. There are two ways to generate NO in plants viz. l-arginine-dependent nitric oxide synthase (NOS) pathway and nitrite-dependent nitrate reductase (NR) pathway. Although NOS-like activity has been detected in plants, this enzyme remains enigmatic. No gene or protein with sequence homology to known mammalian type NOS has been found (Crawford 2006). NR works as a major enzymatic source of NO production in plants. It can convert nitrite to NO in vitro and in vivo (Desikan et al. 2002). In Arabidopsis, NR is encoded by two genes, NIA1 and NIA2, which contribute differently to the synthesis of NO in different tissues.

Another is nonenzymatic conversion of nitrite to NO in the apoplast. NO produced in plants at low concentration may rapidly eliminate lipid peroxyl radicals, alter the species and components of reactive oxygen species (ROS), and block the injuries from ROS, induce the expression of antioxidant genes and the activity of antioxidant enzymes (Lamattina et al. 2003).

3 Effects of NO on Developmental PCD

The effects of NO on developmental PCD have been extensively studied (Table 17.1). Gibberellin (GA)-induced PCD in barley aleurone layers is mediated by ROS, because GA greatly reduces the amount of CAT (catalase) and SOD (superoxide dismutase). NO donors, SNP (sodium nitroprusside) and SNAP (S-nitroso-N-acetylpenicillamine) delay the loss of two enzymes and PCD in barley aleurone layers treated with GA, but stimulate slightly the secretion of α-amylase. It is suggested that NO may be an endogenous modulator of PCD in barley aleurone layers (Beligni et al. 2002).

Table 17.1 Reports of nitric oxide-mediated PCD in plants
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Leaf senescence is a highly coordinated process that involves PCD. Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are poorly known. Kolodziejek et al. (2007) found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order. NO was localized in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. The highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence, and lower concentrations were found during later stages that might suggest that NO plays an inductive role in PCD in leaf senescence.

During the seed development, the cells of the nucleus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nuclear cell degeneration has been characterized as a form of developmental PCD. Lombardi et al. (2010) showed that nucleus PCD is accompanied by a considerable production of both NO and hydrogen peroxide (H2O2), and each of the two molecules is able to induce the production of the other and to cause PCD when applied to a living nucleus. Xylem cells have to be killed so as to facilitate the formation of rigid hollow tubes specialized for water transport. NO is also a key factor regulating PCD and lignification during xylem formation (Neill et al. 2005).

4 Role of NO in Hypersensitive Response

NO plays a crucial role in HR during plant-pathogen interactions. NO and H2O2 function in combination with each other all along HR cell death (Table 17.1).

Administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic guanosine monophosphate (cGMP) and cyclic ADP-ribose, two molecules that can serve as secondary messengers for NO signaling in mammals. Consistent with cGMP acting as a secondary messenger in tobacco, NO treatment induced dramatic and transient increases in endogenous cGMP levels. Unregulated NO levels drive a diffusion limited reaction with O2 − to generate peroxynitrite (ONOO−), which is a mediator of cellular injury in many biological systems but not a mediator of HR. The HR is triggered only by balanced production of NO and reactive oxygen intermediates. Increasing the level of O2 − reduces NO-mediated toxicity. HR is activated after interaction of NO not with O2 − but with H2O2. During the HR, SOD accelerates O2 − dismutation to H2O2 to minimize the loss of NO by reaction with O2 − and to trigger HR through NO/H2O2 cooperation. The rates of production and dismutation of O2 − generated during the oxidative burst play a crucial role in the modulation and integration of NO/H2O2 signaling in the HR (Delledonne et al. 2001). The researches on the kinetics of NO production and hypersensitive cell death showed that NO accumulation contributed to HR. NO was first seen as punctate foci at the cell surface. Subsequent NO accumulation patterns were consistent with NO being an intercellular signal that functions in cell-to-cell spread of the HR (Zhang et al. 2003).

Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and NO are sufficient to induce cell death in Arabidopsis cells independently of ROS. NO-induced cell death is a form of PCD, requiring gene expression, and has a number of characteristics of PCD such as chromatin condensation and caspase-like activity in Arabidopsis cells (Clarke et al. 2000). Phytotoxin fusicoccin induces another form of cell death in sycamore (Acer pseudoplatanus L.) cultured cells, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD (Malerba et al. 2008).

5 Involvement of NO in Abiotic Stress-Induced PCD

NO is also involved in PCD induced by abiotic stress including heat shock, salt, drought, cold, UV radiation, ozone, and heavy metals (mainly cadmium, aluminum) (Table 17.1).

Arabidopsis thaliana cell suspension cultures underwent a PCD process when exposed to 100 and 150 mM CdCl2. As suggested by the expression of the marker senescence-associated gene12 (SAG12), this process resembled an accelerated senescence. CdCl2 treatment was accompanied by a rapid increase in NO and phytochelatin (PC) synthesis, which continued to be high as long as cells remained viable. NO is actually required for Cd2+-induced cell death, because the inhibition of NO synthesis by NG-monomethylarginine monoacetate (l-NMMA) resulted in partial prevention of H2O2 increase, SAG12 expression, and mortality. NO also modulated the extent of PC content and their function by S-nitrosylation (De Michele et al. 2009). Tobacco BY-2 cells exposed to 150 μM CdCl2 underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the PCD, the production of NO increased significantly. NO played a positive role in CdCl2-induced PCD by modulating Cd2+ uptake and thus promoting Cd2+ accumulation in BY-2 cells (Ma et al. 2010). The roots of 3-day-old yellow lupine seedlings exposed to 89 mM CdCl2 resulted in PCD starting from 24 h of stress duration. Cd-induced PCD was preceded by a relatively early burst of NO localized mainly in the root tips. Above changes were accompanied by the NADPH-oxidase-dependent superoxide anion (O .−2 ) production. NADPH-oxidase inhibitor and NO-scavenger significantly reduced O .−2 and NO production, respectively, as well as diminished the pool of cells undergoing PCD (Arasimowicz-Jelonek et al. 2012).

Tobacco leaves, exposed to moderate high light, dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants. The results consolidate significant crosstalk between NO and H2O2, and provide new insight into the early transcriptional response of plants to increased NO and H2O2 levels, and identify target genes of the combined action of NO and H2O2 during the induction of plant cell death (Zago et al. 2006). Lin et al. (2011) identified an NO accumulation mutant noe1 (nitric oxide excess 1) in rice and analyzed its role in NO-mediated leaf cell death. The NOE1, encoded a rice catalase OsCATC, that increased the H2O2 in the leaves, which consequently promoted NO production via activation of NR. Removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death.

Ozone (O3) induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. NO production coincided with the formation of HR-like lesions. SNP and O3 individually induced a large set of defense-related genes; however, in a combined treatment SNP attenuated the O3 induction of salicylic acid (SA) biosynthesis and other defense-related genes. SNP treatment decreased O3-induced SA accumulation. The O3-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 (Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1), a mutant with decreased production of NO, was also O3 sensitive. NO can modify signaling, hormone biosynthesis and gene expression in plants during O3 exposure. NO is an important signaling molecule, which production is needed for a proper O3 response (Ahlfors et al. 2009a, b).

The involvement of cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition of NO and/or ROS generators to the culture medium. The generation of NO did not cause an increase in PAL activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed (De Pinto et al. 2002). Treatment of tobacco protoplasts with SNP resulted in a rapid [Ca2+]cyt accumulation and decrease in mitochondrial membrane potential (potential (ΔΨm) before the appearance of PCD. NO-induced PCD could be largely prevented not only by cPTIO, but also by Ca2+ chelator, EGTA (ethylene glycol tetraacetic acid), Ca2+-channel blocker LaCl3 (Lanthanum chloride) or CsA (a specific mitochondrial permeability transition pore inhibitor, which also inhibit Ca2+ cycling by mitochondria). NO-induced PCD is mediated through mitochondrial pathway and regulated by Ca2+ (Wang et al. 2010a, b). The effects of different NO-donors releasing NO with either NO+ (SNP) or NO− (SNAP, GSNO, NOC-18) character have been compared in plant cells. SNP behaves differently than the other NO-donors tested; indeed, SNP induces accumulation of ferritin transcripts in Arabidopsis, whereas SNAP (S-nitroso-N-acetylpenicillamine) inhibits its accumulation. Only SNP caused PCD and suppression of ROS-scavenging systems (Murgia et al. 2004). Artificial NO donors are widely used as tools to study the role of NO in plants. However, reliable and reproducible characterizations of metabolic responses induced by different NO donors are complicated by the variability of their NO release characteristics. NO release characteristics of the donors SNP, S-nitrosoglutathione (GSNO) and NOS, both in vitro and in planta (Nicotiana tabacum L. cv. BelW3) were evaluated and their effects on NO dependent processes such as the transcriptional regulation of the mitochondrial alternative oxidase (AOX) gene, accumulation of H2O2 and induction of cell death were assessed. Contrary to NOS and SNP, GSNO is not an efficient NO generator in leaf tissue. In spite of the different NO release signatures by SNP and NOS in tissue, the NO-dependent responses examined were similar, suggesting that there is a critical threshold for the NO response (Ederli et al. 2009).

6 Regulation of NO on PCD–Associated Genes Expression

Vacuole processing enzymes (VPEs) are a vacuole-localized cysteine protease, which exhibit caspase-1-like protein activity. It can mediate the activation of caspase-3-like protein to provoke PCD and is involved in virus-induced hypersensitive cell death in tobacco (Hatsugai et al. 2004). VPE activity is also required for aluminum (Al)-induced PCD in plants. Ced-9 inhibited both the Al-induced activity of caspase-like VPE and Al-induced PCD in tobacco (Wang et al. 2009). Senescence-associated gene 12 (SAG12) is considered the best molecular marker of senescence. The expression of SAG12 increased at 2 and 3 d after 100 μM CdCl2 treatment (De Michele et al. 2009). Al-induced PCD was promoted by AhSAG, a senescence-associated gene in Arachis hypoganea (Zhan et al. 2013). As one of the few endogenous cell death inhibitors in plants, bax inhibitor-1 (BI-1) is potentially a core regulator of PCD (Huckelhoven 2004). PpBI-1 can attenuate Al-induced PCD and enhance Al tolerance in transgenic yeast (Zheng et al. 2007). The programmed cell death 5 (PDCD5) gene encodes a protein that shares significant homology with the corresponding proteins of species ranging from yeast to mice (Liu et al. 1999). Overexpression of OsPDCD5 gene induces PCD in rice (Attia et al. 2005).

In tobacco, mechanical wounding induced the rapid transcript accumulation and activation of wound-induced protein kinase (WIPK) (Seo et al. 1995). Transgenic tobacco plants ectopically expressing AhMPK3 exhibited enhanced resistance to first and second instar larvae of Spodoptera litura (Kumar et al. 2009). The conditional overexpression of AhMPK6 resulted in HR-like cell death in tobacco (Kumar and Kirti 2010). MPK kinase 6-mediated activation of VPE modulates heat shock-induced PCD in Arabidopsis (Li et al. 2012). NO promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana PCD (Ye et al. 2012). Over-expression of OsGSNOR reduced intracellular SNO levels, which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants (Lin et al. 2011).

Cytochorme c gets to the cytoplasm at least via two mechanisms. One is via formation of a transient mitochondrial permeability transition pore (MPTP), which is produced by the voltage-dependent anion channel (VDAC) on the outer membrane, the adenine nucleotide transporter (ANT) from the inner membrane and cyclophilin D in the matrix (Green and Reed 1998). Another is directly via the VDAC (Shimizu et al. 1999). Because the expression of AOX, the unique respiratory terminal oxidase in plants, can scavenge excess superoxide anion so that the balance of NO and H2O2 is destroyed, AOX plays protective roles in Al-induced Arabidopsis protoplast death (Li and Xing 2011). As a molecular chaperone, mitochondrial HSP70 may be involved in PCD initiation by reducing Δψm in mitochondrial outer membrane (Chen et al. 2009). Through NO/H2O2 cooperation, SOD accelerates O2 − dismutation to H2O2 to minimize the loss of NO by reaction with O2 − and to trigger hypersensitive cell death (Delledonne et al. 2001). Some genes associated with PCD are listed in Table 17.2.

Table 17.2 Genes in relation to PCD
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7 Interaction Between NO and Other Signaling Molecules During Plant PCD

There are diverse interactions between NO and other signaling molecules such as H2O2, calcium, ethylene, and SA during PCD. The interaction between NO and H2O2 can be cytotoxic or protective. NO/H2O2 cooperation triggers hypersensitive cell death in soybean cell suspensions (Delledonne et al. 2001). Boosted NO and O .−2 production is required for Cd-induced PCD in lupine roots. Moreover, the NO-dependent Cd-induced PCD in roots of 14-day-old lupine plants was correlated with the enhanced level of the post-stress signals in leaves, including distal NO crosstalk with H2O2 (Arasimowicz-Jelonek et al. 2012). Using biochemical and genetic approaches in the root system, Wang et al. (2010a, b) proposed a pathway for the regulation of NO biosynthesis that involves the modulation of NIA2 by MPK6. With the increase of intracellular H2O2 levels, MPK6 is activated, which in turn leads to the phosphorylation of NIA2 at Ser-627. Phosphorylation of NIA2 by MPK6 dramatically.

Increases the activity of NIA2 and the production of NO and also results in morphological changes. SNP treatment resulted in a rapid [Ca2+]cyt accumulation and the appearance of PCD in tobacco protoplasts. EGTA, LaCl3 or CsA largely prevent NO-induced PCD that is mediated through mitochondrial pathway and regulated by Ca2+ (Wang et al. 2010a, b). Moreover, NO is involved in PCD induction via interacting with the pathways of phytohormones (Wang et al. 2010a, b). NO treatments induce ethylene production in tobacco. NO and ethylene act together to regulate O3-induced AOX expression (Ederli et al. 2006). Transcript profiling indicated a role for NO in attenuation of certain classes of O3 induced genes, many of which were related to SA biosynthesis or SA signaling (Ahlfors et al. 2009a, b).

8 NO Signaling Network in Response to PCD

Based on understanding of related knowledge, we propose NO signaling network in response to PCD in plants (Fig. 17.1). Different signals (developmental, pathogen, invasion, and abiotic stress) trigger NO production. Subsequently, NO promotes the expression of PCD-associated genes (such as VPE, AOX, HSP70, APX) via several pathways. One is cGMP-dependent pathway: NO and cGMP mediate the auxin response during adventitious root formation in cucumber (Pagnussat et al. 2003). Moreover, NO regulates the apoptotic signal cascade through protein S-nitrosylation (Wang et al. 2010a, b). Lin et al. (2011) suggested that S-nitrosylation was involved in light-dependent leaf cell death in noe1 rice. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and thioredoxin, which have been reported to be involved in S-nitrosylation regulated cell death in animals. The last one is MAPK cascade. Asai et al. (2002) identified a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2. Signaling events initiated by diverse pathogens converge into a conserved MAPK cascade. An MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway (Pagnussat et al. 2004). NO mediated caspase-3-like protease activation under Cd2+ stress conditions. Pretreatment with cPTIO effectively inhibited Cd2+-induced MAPK activation. Cd2+-induced caspase-3-like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase-3-like protease activation (Ye et al. 2013). NO contributed caspase-3-like protease activation in Cd2+ induced Arabidopsis thaliana PCD, which was mediated by MPK6 (Ye et al. 2012). NO could also regulate the activity of Ca2+-dependent protein kinase (CDPK) was addressed by Lanteri et al. (2006) who characterized a 50 kDa NO-dependent CDPK in cucumber hypocotyls. These three pathways may work synergistic or solely. In turn, gene expressions provoke some downstream events such as PCD.

Fig. 17.1
figure 1

Proposed NO signaling network in response to PCD

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9 Control of NO Level in Plant Mitochondrion

Previous studies showed mitochondrion has emerged as modulating center of plant PCD and also important sites in controlling NO levels in plants. Nitrite (the source of NO synthesis) inhibited the respiration of isolated Arabidopsis mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via AOX or cytochrome c oxidase (COX), respectively. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H2O2 production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. The accumulation of NO derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated Arabidopsis mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells (Wulff et al. 2009). Complex III, COX, and AOX are all involved in nitrite to NO reduction. AOX controls NO generation by directly influencing the rate of electron leakage to nitrite (Cverkovska and Vanlerberghe 2012). Robson and Vanlerberghe (2002) found that knocking down of AOX increases the susceptibility of plants to PCD. There exists a negative feedback loop where NO acts to suppress excess mitochondrial reactive nitrogen species (RNS) and presumably ROS via increased AOX expression to modulate the elicitation of PCD. Three mechanisms of AOX-mediated ROS and RNS homeostasis are suggested. First, AOX can modulate the membrane potential and reduce NO levels. Second, aconitase inhibition leads to increase in citrate which induces AOX to maintain electron flow through the electron transport chain and to lower NO concentrations (Gupta et al. 2012). Third, AOX scavenging of NO might help in decreasing ROS production by preventing over-reduction of ubiquinone pool. However, plant lead to PCD or necrotic cell death in response to stress, because NO and ROS generation from nonmitochondrial sources could swamp any AOX-mediated homeostatic mechanisms.

10 Conclusion and Perspectives

Adverse environmental conditions interferes NO-mediated signal transduction. By direct scavenging of ROS or activating antioxidant enzymes, exogenously applied NO might alleviate metal toxicity in plants. In contrast, NO through S-nitrosylation of PCs or promoting metal uptake via iron transporters contributes or even amplifies metal toxicity. The promoting and suppressing effects of NO on cell death is dependent on a variety of factors, such as cell type, cellular redox status, and the flux and dose of local NO (Wang et al. 2010a, b). Cell signaling dysregulation induced by metal not only leads to the death stimulation pathway, but might be able to activate survival signaling towards tolerance response to heavy metal. Active cell death is required for an enhanced effectiveness of protective responses in neighboring cells (Overmyer et al. 2003). In particular, the relationship between NO, ROS signaling and stress-related hormones might play a key role on the dispute on the expression of gene sets responsible for stress tolerance and in the generation of long-distance sensing from roots to shoots. NO is involved in the generation of systemic signal in systemic acquired resistance to pathogens (Vlot et al. 2008). Xiong et al. (2011) showed that tungstate is not completely a specific NR inhibitor in plant NO research. To investigate the roles of NO in plants, it is necessary to search for more NR-deficient mutants and new specific NR inhibitors. The research on transcriptional factors and NO-regulated genes is the key to understand the mechanism of NO in PCD in higher plants. The recognition of the molecular NO targets will be an exciting challenge for future research.