Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth,Sphingolipid Metabolism,Programmed Cell Death,and Mycotoxin Resistance

Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B₁ (FB₁). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB₁, whereas LOH1-overexpressing plants showed no increase in FB₁ resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB₁. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance for improved plant performance.Ceramides are central intermediates in sphingolipid biosynthesis and mediators of programmed cell death (PCD) in plants (Dunn et al., 2004; Saucedo-García et al., 2011; Ternes et al., 2011a). Ceramides are synthesized by ceramide synthase (or sphingosine N-acyltransferase; EC 2.3.1.24), which catalyzes the formation of an amide linkage between a sphingoid long-chain base (LCB) and a fatty acid using LCB and fatty acyl-CoA substrates (Mullen et al., 2012). The LCB substrate can have two or three hydroxyl groups that are referred to as dihydroxy or trihydroxy LCBs, respectively (Chen et al., 2010). The fatty acyl-CoA substrates typically have chain lengths of C16 or C22 to C26 (Dunn et al., 2004). The latter are referred to as very-long-chain fatty acids (VLCFAs). The ceramide product of ceramide synthase is used primarily as a substrate for the synthesis of either of the two major glycosphingolipids found in plants: glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC; Chen et al., 2010). These glycosphingolipids are major structural components of the plasma membrane and other endomembranes of plant cells (Verhoek et al., 1983; Sperling et al., 2005). In this role, they contribute to membrane physical properties that are important for the ability of plant cells to adjust to environmental extremes and to Golgi-mediated protein trafficking of proteins, including cell wall metabolic enzymes and auxin transporters that underlie plant growth (Borner et al., 2005; Markham et al., 2011; Mortimer et al., 2013; Yang et al., 2013). Alternatively, ceramides can be converted to ceramide-1-phosphates by ceramide kinase activity (Liang et al., 2003). The interchange of ceramides between their free and phosphorylated forms has been linked to the regulation of PCD and PCD-associated resistance to pathogens via the hypersensitive response (HR; Liang et al., 2003; Bi et al., 2014; Simanshu et al., 2014).The Arabidopsis (Arabidopsis thaliana) genome contains three ceramide synthase genes denoted LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540), LOH2 (At3g19260), and LOH3 (At1g13580; Markham et al., 2011; Ternes et al., 2011a). These studies suggest that LOH1 and LOH3 polypeptides are structurally related and catalyze primarily the amidation reaction of trihydroxy LCBs and CoA esters of VLCFAs. The LOH2 polypeptide is more distantly related to LOH1 and LOH3 and catalyzes primarily the condensation of dihydroxy LCBs and C16 fatty acyl-CoAs (Chen et al., 2008; Markham et al., 2011; Ternes et al., 2011a). The ceramide products of LOH1 and LOH3 are most prevalent in GIPC, whereas the ceramide products of LOH2 are more enriched in GlcCer (Markham and Jaworski, 2007; Chen et al., 2008; Ternes et al., 2011b). Similar to plants, the six ceramide synthase isoforms found in humans and mice have distinct specificities for their LCB and acyl-CoA substrates, and these specificities contribute to the formation of complex sphingolipids with differing structures and functions (Venkataraman et al., 2002; Riebeling et al., 2003; Mizutani et al., 2005, 2006; Laviad et al., 2008).In Arabidopsis, LOH1 and LOH3 are partially redundant, but the combined activities of the corresponding polypeptides are essential for plant cell viability, as null double mutants of these genes are lethal (Markham et al., 2011). In contrast, mutants of LOH2 are viable and display no apparent growth phenotype, which brings into question the role of LOH2 ceramide synthase in plant performance (Markham et al., 2011; Ternes et al., 2011a). Overall, these observations indicate that sphingolipids with LOH1-/LOH3-derived trihydroxy LCBs and VLCFA ceramides are essential, but LOH2-derived dihydroxy LCBs and C16 fatty acid ceramides are not required by plant cells. Related to this, LCB C-4 hydroxylase mutants that are deficient in trihydroxy LCBs accumulate elevated amounts of sphingolipids with dihydroxy LCB- and C16 fatty acid-containing ceramides via LOH2 activity (Chen et al., 2008). These mutants are severely impaired in growth and do not transition from vegetative to reproductive growth (Chen et al., 2008).Ceramide synthases are known targets for competitive inhibition by sphingosine analog mycotoxins, including fumonisin B₁ (FB₁) and AAL toxin, produced by pathogenic fungi such as various Fusarium spp. and Alternaria alternata f. sp. lycopersici (Abbas et al., 1994). Inhibition of ceramide synthase results in the accumulation of LCBs that are believed to trigger PCD and result in cytotoxicity (Abbas et al., 1994). In studies of LOH mutants, treatment of Arabidopsis seedlings with FB₁ resulted in not only increases in LCBs but also increases in C16 fatty acid-containing sphingolipids and decreases in VLCFA-containing sphingolipids (Markham et al., 2011; Ternes et al., 2011a). The interpretation of this observation was that FB₁ preferentially inhibits LOH1 and LOH3 ceramide synthases but inhibits LOH2 ceramide synthase to a lesser extent (Markham et al., 2011; Ternes et al., 2011a).Given the findings from Arabidopsis mutants that LOH1 and LOH3 ceramide synthases have distinct substrate specificities and sensitivity to FB₁ relative to LOH2, we hypothesized that the overexpression of each of these ceramide synthases would lead to the production of different sphingolipid compositions as well as different growth phenotypes. This report details experiments designed to test this hypothesis. Among the results presented is a large divergence in the effects of the overexpression of LOH1 and LOH3 versus LOH2 on the growth of Arabidopsis. LOH2 overexpression was also shown to result in sphingolipid compositional, growth, and physiological phenotypes that closely mimic those observed previously in LCB C-4 hydroxylase mutants (Chen et al., 2008).     

Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.Programmed cell death (PCD) is a universal process in multicellular organisms, contributing to the controlled and active degradation of the cell. In plants, PCD is required for processes as diverse as development, self-incompatibility, and stress response. One well-documented example is the induction of PCD upon pathogen attack, allowing the confinement of the infection, and resistance of the plant. The signaling events leading to the onset of PCD have been extensively studied: pathogen recognition triggers activation of mitogen-activated protein kinase cascades, as well as production of reactive oxygen species (ROS) and salicylic acid (SA), which lead to a hypersensitive response (Coll et al., 2011).From a cellular point of view, several classes of plant PCD have been described and compared with the ones found in animal cells (van Doorn, 2011). PCD is thought to have evolved independently in plants and animals, and genes underlying these mechanisms are therefore poorly conserved between the two kingdoms. However, most cellular features are conserved between plant and animal PCD that are both characterized by cell shrinkage, chromatin condensation, DNA laddering, mitochondria permeabilization, and depolarization (Dickman and Fluhr, 2013). In animal cells, mitochondria play a central role in the regulation of apoptosis (Czabotar et al., 2014; Mariño et al., 2014), and this role is likely shared between the two kingdoms (Lord and Gunawardena, 2012). That said, additional mitochondria-independent PCD pathways have clearly evolved in plants.Genetic approaches have greatly contributed to our understanding of cellular pathways governing PCD in plants. For example, the isolation of lesion mimic mutants (LMMs), in which cell death occurs spontaneously, has allowed the identification of several negative regulators of cell death (for review, see Bruggeman et al., 2015b). Interestingly, lesion formation is light dependent in several of these mutants, which include one of the best characterized LMMs—lesions simulating disease1 (lsd1; Dietrich et al., 1994). The LSD1 protein is required for plant acclimation to excess excitation energy (Mateo et al., 2004): when plants are exposed to excessive amounts of light, the redox status of the plastoquinone pool in the chloroplastic electron transfer chain is thought to influence LSD1-dependent signaling to modulate cell death (Mühlenbock et al., 2008). Additionally, we have previously identified the myoinositol phosphate synthase1 (mips1) mutant as a LMM, in which lesion formation is also light dependent (Meng et al., 2009). This mutant is deficient in the myoinositol (MI) phosphate synthase that catalyzes the first committed step of MI biosynthesis and displays pleiotropic defects such as reduced root growth, abnormal vein development, and spontaneous cell death on leaves, together with severe growth reduction after lesions begin to develop (Meng et al., 2009; Donahue et al., 2010). The light-dependent PCD in the mips1 mutant, as observed for lsd1, suggests that chloroplasts may play a role in the MI-dependent cell death regulation. Accumulating evidence suggests that chloroplasts may play a central role in PCD regulation like mitochondria in animal cells (Wang and Bayles, 2013). First, as described in the case of lsd1, excess light energy received by the chloroplast can function as a trigger for PCD. Furthermore, singlet oxygen (¹O₂), a ROS, can activate the EXECUTER1 (EX1) and EX2 proteins in the chloroplasts to initiate PCD (Lee et al., 2007). Likewise, ROS generated by chloroplasts play a major role for PCD onset during nonhost interaction between tobacco (Nicotiana tabacum) and Xanthomonas campestris (Zurbriggen et al., 2009). Finally, functional chloroplasts have also been shown to be required for PCD in cell suspensions (Gutierrez et al., 2014) and in a number of LMMs (Mateo et al., 2004; Meng et al., 2009; Bruggeman et al., 2015b). Thus, chloroplasts are now recognized as important components of plant defense response against pathogens (Stael et al., 2015) and are proposed to function with mitochondria in the execution of PCD (Van Aken and Van Breusegem, 2015). However, the exact signaling and metabolic contribution of chloroplasts to PCD remain to be elucidated. Furthermore, cross talk between chloroplasts and mitochondria does occur, such as during photorespiration (Sunil et al., 2013), but whether such communication functions sequentially or in parallel in the control of PCD remains to be determined (Van Aken and Van Breusegem, 2015).To further investigate how chloroplasts contribute to the regulation of cell death, we performed both forward and reverse genetics on the mips1 mutant. An extragenic secondary mutation in divinyl protochlorophyllide 8-vinyl reductase involved in chlorophyll biosynthesis leads to chlorophyll deficiency that abolishes the mips1 cell death phenotype, as do changes in CO₂ availability. These findings provide evidence for a link between photosynthetic activity and PCD induction in mips1. Additionally, we investigated the contribution of several retrograde signaling pathways (Chan et al., 2015) to the control of PCD in mips1. This process was independent of GENOMES UNCOUPLED (GUN) and EX signaling pathways, but we found that the SAL1-PAP_XRN retrograde signaling pathway inhibits cell death as well as basal defense reactions in Arabidopsis (Arabidopsis thaliana).     

S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H₂O₂) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H₂O₂-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H₂O₂-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H₂O₂-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H₂O₂ levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.Nitric oxide (NO) is a gaseous and diffusible redox molecule that acts as a signaling compound in both animal and plant systems (Pacher et al., 2007; Besson-Bard et al., 2008). In plants, NO has been found to play a key role in several physiological processes, such as germination, lateral root development, flowering, senescence, stomatal closure, and growth of pollen tubes (Beligni and Lamattina, 2000; Neill et al., 2002; Correa-Aragunde et al., 2004; He et al., 2004; Prado et al., 2004; Carimi et al., 2005). In addition, NO has been reported to be involved in plant responses to both biotic and abiotic stresses (Leitner et al., 2009; Siddiqui et al., 2011) and in the signaling pathways leading to programmed cell death (PCD; Delledonne et al., 1998; de Pinto et al., 2006; De Michele et al., 2009; Lin et al., 2012; Serrano et al., 2012).The cellular environment may greatly influence the chemical reactivity of NO, giving rise to different biologically active NO-derived compounds, collectively named reactive nitrogen species, which amplify and differentiate its ability to activate physiological and stress-related processes. Many of the biological properties of NO are due to its high affinity with transition metals of metalloproteins as well as its reactivity with reactive oxygen species (ROS; Hill et al., 2010). However, recent evidence suggests that protein S-nitrosylation, due to the addition of NO to reactive Cys thiols, may act as a key mechanism of NO signaling in plants (Wang et al., 2006; Astier et al., 2011). NO is also able to react with reduced glutathione (GSH), the most abundant cellular thiol, thus producing S-nitrosoglutathione (GSNO), which also acts as an endogenous trans-nitrosylating agent. GSNO is also considered as a NO store and donor and, as it is more stable than NO, acts as a long-distance NO transporter through the floematic flux (Malik et al., 2011). S-Nitrosoglutathione reductase (GSNOR), which is an enzyme conserved from bacteria to humans, has been suggested to play a role in regulating S-nitrosothiols (SNO) and the turnover of S-nitrosylated proteins in plants (Liu et al., 2001; Rusterucci et al., 2007).A number of proteins involved in metabolism, stress responses, and redox homeostasis have been identified as potential targets for S-nitrosylation in Arabidopsis (Arabidopsis thaliana; Lindermayr et al., 2005). During the hypersensitive response (HR), 16 proteins were identified to be S-nitrosylated in the seedlings of the same species (Romero-Puertas et al., 2008); in Citrus species, S-nitrosylation of about 50 proteins occurred in the NO-mediated resistance to high salinity (Tanou et al., 2009).However, while the number of candidate proteins for S-nitrosylation is increasing, the functional significance of protein S-nitrosylation has been explained only in a few cases, such as for nonsymbiotic hemoglobin (Perazzolli et al., 2004), glyceraldehyde 3-phosphate dehydrogenase (Lindermayr et al., 2005; Wawer et al., 2010), Met adenosyltransferase (Lindermayr et al., 2006), and metacaspase9 (Belenghi et al., 2007). Of particular interest are the cases in which S-nitrosylation involves enzymes controlling ROS homeostasis. For instance, it has been reported that S-nitrosylation of peroxiredoxin IIE regulates the antioxidant function of this enzyme and might contribute to the HR (Romero-Puertas et al., 2007). It has also been shown that in the immunity response, S-nitrosylation of NADPH oxidase inactivates the enzyme, thus reducing ROS production and controlling HR development (Yun et al., 2011).Recently, S-nitrosylation has also been shown to be involved in PCD of nitric oxide excess1 (noe1) rice (Oryza sativa) plants, which are mutated in the OsCATC gene coding for catalase (Lin et al., 2012). In these plants, which show PCD-like phenotypes under high-light conditions, glyceraldehyde 3-phosphate dehydrogenase and thioredoxin are S-nitrosylated. This suggests that the NO-dependent regulation of these proteins is involved in plant PCD, similar to what occurs in animal apoptosis (Sumbayev, 2003; Hara et al., 2005; Lin et al., 2012). The increase in hydrogen peroxide (H₂O₂) after exposure to high light in noe1 plants is responsible for the production of NO required for leaf cell death induction (Lin et al., 2012). There is a strict relationship between H₂O₂ and NO in PCD activation (Delledonne et al., 2001; de Pinto et al., 2002); however, the mechanism of this interplay is largely still unknown (for review, see Zaninotto et al., 2006; Zhao, 2007; Yoshioka et al., 2011). NO can induce ROS production and vice versa, and their reciprocal modulation in terms of intensity and timing seems to be crucial in determining PCD activation and in controlling HR development (Delledonne et al., 2001; Zhao, 2007; Yun et al., 2011).In previous papers, we demonstrated that heat shock (HS) at 55°C and treatment with 50 mm H₂O₂ promote PCD in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Vacca et al., 2004; de Pinto et al., 2006; Locato et al., 2008). In both experimental conditions, NO production and decrease in cytosolic ascorbate peroxidase (cAPX) were observed as early events in the PCD pathway, and cAPX decrease has been suggested to contribute to determining the redox environment required for PCD (de Pinto et al., 2006; Locato et al., 2008).In this study, the production of nitrosylating agents (NO and GSNO) in the first hours of PCD induction by HS or H₂O₂ treatment in tobacco BY-2 cells and their role in PCD were studied. The possibility that S-nitrosylation could be a first step in regulating cAPX activity and turnover as part of the signaling pathway leading to PCD was also investigated.     

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]_cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]_cyt of the pollen tubes in vivo, we show that [pH]_cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]_cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]_cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]_cyt and its effect on several components in the context of SI-induced events and PCD.Programmed cell death (PCD) in plants is relatively well documented and characterized (Jones and Dangl, 1996; van Doorn, 2011; van Doorn et al., 2011). There is considerable biochemical evidence for the involvement of caspase-like activities in plant PCD (van Doorn and Woltering, 2005). For example, the vacuolar processing enzyme has YVADase (caspase-1-like) activity (Hatsugai et al., 2004; Rojo et al., 2004; Hara-Nishimura et al., 2005), DEVDase (caspase-3-like) and YVADases are associated with PCD in several plant systems (del Pozo and Lam, 1998; Korthout et al., 2000; Danon et al., 2004), and VEIDase (caspase-6-like) is the main caspase-like activity involved in embryonic pattern formation (Bozhkov et al., 2004). However, because plants have no caspase gene homologs (Sanmartín et al., 2005), the nature of their caspase-like enzymes is the subject of considerable debate. Vacuolar cell death is one of two major classes of PCD in plants (van Doorn et al., 2011). It is thought that collapse of the vacuole is a key irreversible step in several plant PCD systems, including during tissue and organ formation, such as the classic differentiation of tracheary elements (Hara-Nishimura and Hatsugai, 2011). Exactly how this is achieved and what processes are involved remain unknown. Until very recently, it was generally thought that the rupturing vacuole releases proteases, hydrolases, and nucleases, allowing cellular disassembly by an autophagy-like process. Some PCD systems cannot be assigned to either class; these include PCD triggered by the hypersensitive response to biotrophic pathogens, PCD in cereal endosperm, and self-incompatibility (SI)-induced PCD (van Doorn et al., 2011).SI is a genetically controlled pollen-pistil cell-cell recognition system. Self-pollen is recognized by the stigma as being genetically identical, resulting in inhibition of pollen tube growth. Most SI systems use tightly linked polymorphic genes: the pollen (male) and pistil (female) S-determinants. In field poppy (Papaver rhoeas), the S-determinants are a 14-kD signaling ligand field poppy stigma S (PrsS) and a unique transmembrane protein field poppy pollen S (PrpS; Foote et al., 1994; Wheeler et al., 2010). These interact in an S-specific manner, and increases in cytosolic free calcium ([Ca2+]_cyt) are triggered in incompatible pollen tubes (Franklin-Tong et al., 1993), resulting in phosphorylation of soluble inorganic pyrophosphatases (sPPases; Rudd et al., 1996; de Graaf et al., 2006), activation of a Mitogen-Activated Protein Kinase (MAPK; Rudd et al., 2003), and increases in reactive oxygen species (ROS) and nitric oxide (Wilkins et al., 2011, 2014). Most of these components are integrated into a signaling network leading to PCD (Bosch et al., 2008; Wilkins et al., 2014). The actin cytoskeleton is a key target in the field poppy SI response, undergoing depolymerization (Snowman et al., 2002) followed by polymerization into highly stable F-actin foci decorated with the actin binding proteins (ABPs) Actin-Depolymerizing Factor (ADF) and Cyclase-Associated Protein (CAP; Poulter et al., 2010, 2011), with both processes being involved in mediating PCD (Thomas et al., 2006). A major player in SI-mediated PCD is a caspase-3-like/DEVDase-like activity (Thomas and Franklin-Tong, 2004; Bosch and Franklin-Tong, 2007). The SI-induced caspase-3-like/DEVDase exhibits maximum substrate cleavage in vitro at pH 5, with peak activity 5 h after SI induction in vivo (Bosch and Franklin-Tong, 2007). The low pH optimum for this caspase-3-like/DEVDase activity is unusual, because most of the cytosolic plant caspase-like activities identified to date have optimal activity close to normal physiological pH (approximate pH, 6.5–7.0; Korthout et al., 2000; Bozhkov et al., 2004; Coffeen and Wolpert, 2004). Because the SI-induced cytosolic-located DEVDase requires a low pH for activity, this suggested that, during SI, the pollen tube cytosol undergoes dramatic acidification. In vivo pH measurements of the cytosol at 1 to 4 h after SI induction confirmed this, when cytosolic pH ([pH]_cyt) had dropped from pH 6.9 to pH 5.5 (Bosch and Franklin-Tong, 2007). This fits the in vitro pH optimum of the caspase-3-like/DEVDase almost exactly, implicating pollen cytosolic acidification as playing a vital role in creating optimal conditions for the activation of the caspase-3-like/DEVDase-like activity and progression of PCD.Under normal cellular conditions, [pH]_cyt is between approximately 6.9 and 7.5 (Kurkdjian and Guern, 1989; Felle, 2001). Pollen tubes, like other tip-growing cells, have [pH]_cyt gradients (Gibbon and Kropf, 1994; Feijó et al., 1999). The [pH]_cyt of the pollen tube shank is an approximate pH of 6.9 to 7.11 (Fricker et al., 1997; Messerli and Robinson, 1998). There has been much debate about the [pH]_cyt gradient, comprising an apical domain with an approximate pH of 6.8 and a subapical alkaline band with an approximate pH of 7.2 to 7.8 in Lilium longiflorum and Lilium formosanum pollen tubes (Fricker et al., 1997; Messerli and Robinson, 1998; Feijó et al., 2001; Lovy-Wheeler et al., 2006). Oscillations of [pH]_cyt between approximate pH values of 6.9 and 7.3 have been linked to tip growth in L. formosanum pollen tubes (Lovy-Wheeler et al., 2006). The vacuole and the apoplast have a highly acidic pH between pH 5 and pH 6 (Katsuhara et al., 1989; Feijó et al., 1999). The majority of studies of pH changes in plant cells reports modest, transient changes in [pH]_cyt of approximately 0.4 and 0.7 pH units during development, gravitropic responses, decreases in light intensity, and addition of elicitors, hormones, and other treatments. For example, during root hair development in Arabidopsis (Arabidopsis thaliana), root [pH]_cyt was elevated from an approximate pH of 7.3 to 7.7 (Bibikova et al., 1998). Root gravitropic responses stimulate small transient [pH]_cyt alterations (Scott and Allen, 1999; Fasano et al., 2001; Johannes et al., 2001). More recently, it has been shown that the [pH]_cyt drops during PCD controlling root cap development; however, exactly how many units the [pH]_cyt decreased was not measured (Fendrych et al., 2014). Other studies investigating [pH]_cyt in response to physiologically relevant signals also report small transient alterations. Light-adapted cells respond to a decrease in light intensity with a rapid transient cytosolic acidification by approximately 0.3 pH units (Felle et al., 1986). Addition of nodulation factors resulted in an increase of 0.2 pH units in root hairs (Felle et al., 1998), and abscisic acid increased the [pH]_cyt of guard cells by 0.3 pH units (Blatt and Armstrong, 1993). Changes in [pH]_cyt are thought to activate stress responses (Felle, 2001). Elicitor treatments resulted in a [pH]_cyt drop of between 0.4 and 0.7 pH units in suspension cells (Mathieu et al., 1996; Kuchitsu et al., 1997), a drop of 0.2 pH units in Nitellopsis obtusa cells treated with salt (Katsuhara et al., 1989), and a drop of 0.3 to 0.7 pH units in Eschscholzia californica (Roos et al., 1998).Here, we investigate SI-induced acidification of the cytosol, providing measurements of physiologically relevant temporal alterations in [pH]_cyt, and identify key targets of this, providing mechanistic insights into these events. The SI-induced acidification plays a pivotal role in the activation of a caspase-3-like/DEVDase activity, the formation of punctate F-actin foci, and ABP localization during SI PCD. We investigate the vacuole as a potential contributor to SI-induced [pH]_cyt acidification.     

Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.Programmed cell death (PCD) has been defined as a sequence of genetically regulated events that lead to the elimination of specific cells, tissues, or whole organs (Lockshin and Zakeri, 2004). In plants, PCD is essential for developmental processes and defense responses (Dangl et al., 1996; Greenberg, 1996; Durrant et al., 2007). One well-characterized example of plant PCD is the hypersensitive response occurring during incompatible plant-pathogen interactions (Lam, 2004), which results in cell death to form visible lesions at the site of infection by an avirulent pathogen and consequently limits the pathogen spread (Morel and Dangl, 1997).To date, a large number of mutants that display spontaneous cell death lesions have been identified in barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana; Marchetti et al., 1983; Wolter et al., 1993; Dietrich et al., 1994; Gray et al., 1997). Because lesions form in the absence of pathogen infection, these mutants have been collectively termed as lesion-mimic mutants. Many genes with regulatory roles in PCD and defense responses, including LESION SIMULATING DISEASE1, ACCELERATED CELL DEATH11, and VASCULAR ASSOCIATED DEATH1, have been cloned and characterized (Dietrich et al., 1997; Brodersen et al., 2002; Lorrain et al., 2004).The appearance of spontaneous cell death lesions in some lesion-mimic mutants is dependent on photoperiod. For example, the Arabidopsis mutant lesion simulating disease1 and myoinositol-1-phosphate synthase1 show lesions under long days (LD; Dietrich et al., 1994; Meng et al., 2009), whereas the lesion simulating disease2, lesion initiation1, enhancing RPW8-mediated HR-like cell death1, and lag one homolog1 display lesions under short days (SD; Dietrich et al., 1994; Ishikawa et al., 2003; Wang et al., 2008; Ternes et al., 2011).Blockage of some metabolic pathways in plants may cause cell death and result in lesion formation. For example, the lesion-mimic phenotypes in the Arabidopsis mutants lesion initiation2 and accelerated cell death2 and the maize mutant lesion mimic22 result from an impairment of porphyrin metabolism (Hu et al., 1998; Ishikawa et al., 2001; Mach et al., 2001). Deficiency in fatty acid, sphingolipid, and myoinositol metabolism also causes cell death in Arabidopsis (Mou et al., 2000; Liang et al., 2003; Wang et al., 2008; Meng et al., 2009; Donahue et al., 2010; Berkey et al., 2012).Tyr degradation is an essential five-step pathway in animals (Lindblad et al., 1977). First, Tyr aminotransferase catalyzes the conversion of Tyr into 4-hydroxyphenylpyruvate, which is further transformed into homogentisate by 4-hydroxyphenylpyruvate dioxygenase. Through the sequential action of homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI), and fumarylacetoacetate hydrolase (FAH), homogentisate is catalyzed to generate fumarate and acetoacetate (Lindblad et al., 1977). Blockage of this pathway in animals results in metabolic disorder diseases (Lindblad et al., 1977; Ruppert et al., 1992; Grompe et al., 1993). For example, human FAH deficiency causes hereditary tyrosinemia type I (HT1), an inborn lethal disease (St-Louis and Tanguay, 1997). Although the homologous genes putatively encoding these enzymes exist in plants (Dixon et al., 2000; Lopukhina et al., 2001; Dixon and Edwards, 2006), it is unclear whether this pathway is essential for plant growth and development.In this study, we report the isolation and characterization of a recessive short-day sensitive cell death1 (sscd1) mutant in Arabidopsis. Map-based cloning of the corresponding gene revealed that SSCD1 encodes the Arabidopsis putative FAH. Further knockout of the gene encoding the Arabidopsis putative HGO completely eliminated the spontaneous cell death phenotype in the sscd1 mutant. Furthermore, we found that treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway (Lindblad et al., 1977), is able to mimic the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under SD.     

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis

A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD.In response to the constant attack by microbial pathogens, plants have developed defense mechanisms to protect themselves against harmful diseases caused by various pathogens. Plants primarily rely on two layers of innate immunity to cope with microbial pathogens (Jones and Dangl, 2006). The first layer of plant immunity, which is triggered by pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin, lipopolysaccharides, and fungal chitin, is designated PAMP-triggered immunity (PTI; Boller and He, 2009). Because pathogens have evolved to overcome PTI, plants have developed a second layer of immunity, referred to as effector-triggered immunity (ETI; Dodds and Rathjen, 2010). ETI depends on specific interactions between plant Resistance proteins and pathogen effectors and is often associated with a form of programmed cell death (PCD) termed the hypersensitive response (HR), which inhibits pathogen growth (Coll et al., 2011).Plants use PCD to regulate developmental and defense responses. In addition to pathogen attack, many abiotic stress factors such as heat and ozone exposure elicit PCD in plants (Hayward and Dinesh-Kumar, 2011). PCD also occurs during various developmental processes, including endosperm development, tracheary element (TE) differentiation, female gametophyte differentiation, leaf abscission, and senescence (Kuriyama and Fukuda, 2002; Gunawardena, 2008). Recently, plant PCD has been classified into two types, “autolytic” PCD and “nonautolytic” PCD, on the basis of the presence or absence of rapid cytoplasm clearance after tonoplast rupture, respectively (van Doorn et al., 2011). Autolytic PCD, which mainly occurs during plant development, falls under “autophagic” PCD in animals because it is associated with the accumulation of autophagy-related structures in the cytoplasm. Some forms of HR PCD classified as nonautolytic PCD in plants are accompanied by increased vacuolization, indicating the progress of autophagy, and therefore can be placed under autophagic PCD (Hara-Nishimura et al., 2005; Hatsugai et al., 2009).Autophagy is an intracellular process in which double membrane-bound autophagosomes enclose cytoplasmic components and damaged or toxic materials and target them to the vacuole or lysosome for degradation (Chung, 2011). In plants, autophagy plays important roles in the responses to nutrient starvation, senescence, and abiotic and biotic stresses (Liu et al., 2005; Xiong et al., 2005, 2007; Bassham, 2007; Hofius et al., 2009). Accumulating evidence indicates that autophagy regulates immune responses in both animals and plants. Autophagy is essential for the direct elimination of pathogens in mammalian systems (Levine et al., 2011). Invading bacteria and viruses are targeted to autophagosomes and then delivered to the lysosome for degradation in a process called xenophagy (Levine, 2005). In addition to its function in directly killing pathogens, xenophagic degradation can provide microbial antigens for major histocompatibility complex class II presentation to the innate and adaptive immune systems (Levine, 2005; Schmid and Münz, 2007). Furthermore, the human surface receptor CD46 was shown to directly induce autophagy through physical interaction with the autophagic machinery (Joubert et al., 2009). The role of autophagy in plant basal immunity to virulent pathogens has been determined (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009; Lai et al., 2011; Lenz et al., 2011). Arabidopsis (Arabidopsis thaliana) plants defective in AUTOPHAGY-RELATED (ATG) genes exhibited enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, suggesting that the massive breakdown of cytoplasmic materials provides nutrients for the growth of necrotrophic pathogens or that fungal toxin-induced necrotic cell death is enhanced in atg mutants (Lai et al., 2011; Lenz et al., 2011). However, studies on the responses to the biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000) have yielded contradictory results. Whereas earlier studies reported that bacterial numbers significantly increased in ATG6-antisense (AS) and atg mutant plants (Patel and Dinesh-Kumar, 2008; Hofius et al., 2009), a recent study indicated that atg mutants exhibit increased resistance to Pst DC3000 (Lenz et al., 2011). Although these discrepancies remain to be resolved, salicylic acid (SA) levels and SA-dependent gene expression were both elevated in atg mutants, suggesting that autophagy may negatively regulate SA-associated plant immunity (Yoshimoto et al., 2009; Lenz et al., 2011). These findings indicate that the role of autophagy in plant immunity depends on the lifestyle of the invading pathogens (Lenz et al., 2011).Autophagy plays an important role in the regulation of HR PCD in plant innate immunity (Hayward and Dinesh-Kumar, 2011). Tobacco (Nicotiana tabacum) plants silenced for ATG6/Beclin1 and other ATG genes such as phosphatidylinositol 3-kinase (PI3K)/vacuolar protein sorting34 (VPS34), ATG3, and ATG7 underwent unrestricted HR PCD upon pathogen infection (Liu et al., 2005). ATG6-AS and atg5 mutant Arabidopsis plants also displayed unlimited HR PCD upon infection with the avirulent bacterium Pst DC3000 (AvrRpm1; Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009). These studies suggest that autophagy is a “prosurvival” or “antideath” mechanism that negatively regulates HR PCD (Liu and Bassham, 2012). By contrast, a “prodeath” role has been suggested for autophagy in HR PCD regulation (Hofius et al., 2009). Pst DC3000 (AvrRps4)-induced and, to a lesser extent, Pst DC3000 (AvrRpm1)-induced HR PCD was suppressed in atg mutants, suggesting that autophagy plays a positive role and that autophagic cell death is involved in RPS4- and RPM1-mediated HR cell death.We previously showed that the small GTP-binding protein RabG3b, isolated from secretome analysis in Arabidopsis (Oh et al., 2005), functions as a component of autophagy and positively regulates TE differentiation via the activation of autophagic cell death (Kwon et al., 2010a, 2010b). Overexpression of a constitutively active RabG3b (RabG3bCA) in plants significantly increased autophagy during PCD associated with TE differentiation, thereby enhancing TE formation and xylem development. Transgenic poplar (Populus alba × Populus tremula var glandulosa) overexpressing Arabidopsis RabG3bCA was further generated, and these exhibited significant stimulation of xylem development together with autophagic activation, suggesting that RabG3b is a positive regulator of autophagy and xylem development in Populus spp. as well as Arabidopsis (Kwon et al., 2011). We also reported that RabG3b is involved in cell death associated with the fungal pathogen A. brassicicola and infection with the fungal toxin fumonisin B1 (FB1) as well as leaf senescence (Kwon et al., 2009). Here, we extend our work to determine the role of RabG3b and autophagy in immunity-associated HR PCD. We found that RabG3bCA transgenic plants accumulated a large number of autophagic structures and displayed accelerated, expanded cell death against a number of PCD inducers, such as FB1 and the bacterial pathogens Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Our results suggest that RabG3b plays a positive role in immunity-associated HR PCD via the activation of autophagic cell death.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants

Lipid peroxide-derived toxic carbonyl compounds (oxylipin carbonyls), produced downstream of reactive oxygen species (ROS), were recently revealed to mediate abiotic stress-induced damage of plants. Here, we investigated how oxylipin carbonyls cause cell death. When tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells were exposed to hydrogen peroxide, several species of short-chain oxylipin carbonyls [i.e. 4-hydroxy-(E)-2-nonenal and acrolein] accumulated and the cells underwent programmed cell death (PCD), as judged based on DNA fragmentation, an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei, and cytoplasm retraction. These oxylipin carbonyls caused PCD in BY-2 cells and roots of tobacco and Arabidopsis (Arabidopsis thaliana). To test the possibility that oxylipin carbonyls mediate an oxidative signal to cause PCD, we performed pharmacological and genetic experiments. Carnosine and hydralazine, having distinct chemistry for scavenging carbonyls, significantly suppressed the increase in oxylipin carbonyls and blocked PCD in BY-2 cells and Arabidopsis roots, but they did not affect the levels of ROS and lipid peroxides. A transgenic tobacco line that overproduces 2-alkenal reductase, an Arabidopsis enzyme to detoxify α,β-unsaturated carbonyls, suffered less PCD in root epidermis after hydrogen peroxide or salt treatment than did the wild type, whereas the ROS level increases due to the stress treatments were not different between the lines. From these results, we conclude that oxylipin carbonyls are involved in the PCD process in oxidatively stressed cells. Our comparison of the ability of distinct carbonyls to induce PCD in BY-2 cells revealed that acrolein and 4-hydroxy-(E)-2-nonenal are the most potent carbonyls. The physiological relevance and possible mechanisms of the carbonyl-induced PCD are discussed.In plants, environmental stressors such as extreme temperatures, drought, intense UV-B radiation, and soil salinity can cause tissue damage, growth inhibition, and even death. These detrimental effects are often ascribed to the action of reactive oxygen species (ROS) produced in the stressed plants for the following reasons: (1) various environmental stressors commonly cause the oxidation of biomolecules in plants; and (2) transgenic plants with enhanced antioxidant capacities show improved tolerance to environmental stressors (Suzuki et al., 2014). The production of ROS such as superoxide anion radical and hydrogen peroxide (H₂O₂) is intrinsically associated with photosynthesis and respiration (Foyer and Noctor, 2003; Asada, 2006).Plant cells are equipped with abundant antioxidant molecules such as α-tocopherol, β-carotene, and ascorbic acid and an array of ROS-scavenging enzymes such as superoxide dismutase and ascorbate peroxidase to maintain low intracellular ROS levels. When plants are exposed to severe and prolonged environmental stress, the balance between the production and scavenging of ROS is disrupted and the cellular metabolism reaches a new state of higher ROS production and lower antioxidant capacity. Then, the oxidation of vital biomolecules such as proteins and DNA proceeds, and as a consequence, cells undergo oxidative injury (Mano, 2002). The cause-effect relationship between ROS and tissue injury in plants is thus widely accepted, but the biochemical processes between the generation of ROS and cell death are poorly understood.Increasing evidence shows that oxylipin carbonyls mediate the oxidative injury of plants (Yamauchi et al., 2012; for review, see Mano, 2012; Farmer and Mueller, 2013). Oxylipin carbonyls are a group of carbonyl compounds derived from oxygenated lipids and fatty acids. The production of oxylipin carbonyls in living cells is explained as follows. Lipids in the membranes are constitutively oxidized by ROS to form lipid peroxides (LOOHs; Mène-Saffrané et al., 2007) because they are the most immediate and abundant targets near the ROS production sites. There are two types of LOOH formation reaction from ROS (Halliwell and Gutteridge, 2007). One is the radical-dependent reaction. Highly oxidizing radicals, such as hydroxyl radical (standard reduction potential of the HO^•/H₂O pair, +2.31 V) and the protonated form of superoxide radical (HO₂/H₂O₂, +1.06 V), can abstract a hydrogen atom from a lipid molecule, especially at the central carbon of a pentadiene structure in a polyunsaturated fatty acid, to form a radical. This organic radical rapidly reacts with molecular oxygen, forming a lipid hydroperoxyl radical, which then abstracts a hydrogen atom from a neighboring molecule and becomes a LOOH. The other reaction is the addition of singlet oxygen to a double bond of an unsaturated fatty acid to form an endoperoxide or a hydroperoxide (both are LOOHs). A variety of LOOH species are formed, depending on the source fatty acid and also by the oxygenation mechanism (Montillet et al., 2004). LOOH molecules are unstable, and in the presence of redox catalysts such as transition metal ions or free radicals, they decompose to form various aldehydes and ketones (i.e. oxylipin carbonyls; Farmer and Mueller, 2013). The chemical species of oxylipin carbonyl formed in the cells differ according to the fatty acids and the type of ROS involved (Grosch, 1987; Mano et al., 2014a).More than a dozen species of oxylipin carbonyls are formed in plants (for review, see Mano et al., 2009). Oxylipin carbonyls are constitutively formed in plants under normal physiological conditions, and the levels of certain types of oxylipin carbonyls rise severalfold under stress conditions, detected as increases in the free carbonyl content (Mano et al., 2010; Yin et al., 2010; Kai et al., 2012) and by the extent of the carbonyl modification of target proteins (Winger et al., 2007; Mano et al., 2014b). Among the oxylipin carbonyls, the α,β-unsaturated carbonyls, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), have high reactivity and cytotoxicity (Esterbauer et al., 1991; Alméras et al., 2003). They strongly inactivate lipoate enzymes in mitochondria (Taylor et al., 2002) and thiol-regulated enzymes in chloroplasts (Mano et al., 2009) in vitro and cause tissue injury in leaves when they are fumigated (Matsui et al., 2012).The physiological relevance of oxylipin carbonyls has been shown by the observation that the overexpression of different carbonyl-scavenging enzymes commonly confers stress tolerance to transgenic plants (for review, see Mano, 2012). For example, 2-alkenal reductase (AER)-overproducing tobacco (Nicotiana tabacum) showed tolerance to aluminum (Yin et al., 2010), aldehyde dehydrogenase-overproducing Arabidopsis (Arabidopsis thaliana) showed tolerance to osmotic and oxidative stress (Sunkar et al., 2003), and aldehyde reductase-overproducing tobacco showed tolerance to chemical and drought stress (Oberschall et al., 2000). In addition, the genetic suppression of a carbonyl-scavenging enzyme made plants susceptible to stressors (Kotchoni et al., 2006; Shin et al., 2009; Yamauchi et al., 2012; Tang et al., 2014). Under stress conditions, there are positive correlations between the levels of certain carbonyls and the extent of tissue injury (Mano et al., 2010; Yin et al., 2010; Yamauchi et al., 2012). Thus, it is evident that oxylipin carbonyls, downstream products of ROS, are causes of oxidative damage in plant cells.To investigate how oxylipin carbonyls damage cells in oxidatively stressed plants, we here examined the mode of cell death that is induced by oxylipin carbonyls and identified the carbonyl species responsible for the cell death. We observed that oxylipin carbonyls cause programmed cell death (PCD), and our results demonstrated that the oxylipin carbonyls mediate the oxidative stress-induced PCD in tobacco Bright Yellow-2 (BY-2) cultured cells and in roots of tobacco and Arabidopsis plants. We then estimated the relative strengths of distinct carbonyl species to initiate the PCD program. Our findings demonstrate a critical role of the lipid metabolites in ROS signaling.