oiwa,a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase,Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.     

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).     

Ca2+-Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.     

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension cultures (Liang et al., 2003; Lachaud et al., 2010, 2011; Alden et al., 2011) or in whole seedlings (Shi et al., 2007; Takahashi et al., 2009; Saucedo-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or l-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010; Ternes et al., 2011). Deciphering of Cer participation in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011). Furthermore, blocking the conversion of LCBs to LCB-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thus established a link between JA signaling, PCD, and sphingolipid metabolism.     

From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO₂ concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.     

A Wheat SIMILAR TO RCD-ONE Gene Enhances Seedling Growth and Abiotic Stress Resistance by Modulating Redox Homeostasis and Maintaining Genomic Integrity

Plant growth inhibition is a common response to salinity. Under saline conditions, Shanrong No. 3 (SR3), a bread wheat (Triticum aestivum) introgression line, performs better than its parent wheat variety Jinan 177 (JN177) with respect to both seedling growth and abiotic stress tolerance. Furthermore, the endogenous reactive oxygen species (ROS) was also elevated in SR3 relative to JN177. The SR3 allele of sro1, a gene encoding a poly(ADP ribose) polymerase (PARP) domain protein, was identified to be crucial for both aspects of its superior performance. Unlike RADICAL-INDUCED CELL DEATH1 and other Arabidopsis thaliana SIMILAR TO RCD-ONE (SRO) proteins, sro1 has PARP activity. Both the overexpression of Ta-sro1 in wheat and its heterologous expression in Arabidopsis promote the accumulation of ROS, mainly by enhancing the activity of NADPH oxidase and the expression of NAD(P)H dehydrogenase, in conjunction with the suppression of alternative oxidase expression. Moreover, it promotes the activity of ascorbate-GSH cycle enzymes and GSH peroxidase cycle enzymes, which regulate ROS content and cellular redox homeostasis. sro1 is also found to be involved in the maintenance of genomic integrity. We show here that the wheat SRO has PARP activity; such activity could be manipulated to improve the growth of seedlings exposed to salinity stress by modulating redox homeostasis and maintaining genomic stability.     

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.     

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

The signaling role of hydrogen gas (H₂) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H₂ in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H₂, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K⁺ channel (GORK). ABA elicited a rapid and sustained H₂ release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H₂ production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H₂-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.Stomata are responsible for leaves of terrestrial plants taking in carbon dioxide for photosynthesis and likewise regulate how much water plants evaporate through the stomatal pores (Chaerle et al., 2005). When experiencing water-deficient conditions, surviving plants balance photosynthesis with controlling water loss through the stomatal pores, which relies on turgor changes by pairs of highly differentiated epidermal cells surrounding the stomatal pore, called the guard cells (Haworth et al., 2011; Loutfy et al., 2012).Besides the characterization of the significant roles of abscisic acid (ABA) in regulating stomatal movement, the key factors in guard cell signal transduction have been intensively investigated by performing forward and reverse genetics approaches. For example, both reactive oxygen species (ROS) and nitric oxide (NO) have been identified as vital intermediates in guard cell ABA signaling (Bright et al., 2006; Yan et al., 2007; Suzuki et al., 2011; Hao et al., 2012). The key ROS-producing enzymes in Arabidopsis (Arabidopsis thaliana) guard cells are the respiratory burst oxidase homologs (Rboh) D and F (Kwak et al., 2003; Bright et al., 2006; Mazars et al., 2010; Marino et al., 2012). Current available data suggest that there are at least two distinct pathways responsible for NO synthesis involved in ABA signaling in guard cells: the nitrite reductase (NR)- and l-Arg-dependent pathways (Desikan et al., 2002; Besson-Bard et al., 2008). Genetic evidence further demonstrated that removal of the major known sources of either ROS or NO significantly impairs ABA-induced stomatal closure. ABA fails to induce ROS production in the atrbohD/F double mutant (Kwak et al., 2003; Wang et al., 2012) and NO synthesis in the NR-deficient mutant nitrate reductase1/2 (nia1/2; Bright et al., 2006; Neill et al., 2008), both of which lead to impaired stomatal closure in Arabidopsis. Most importantly, ROS and NO, which function both synergistically and independently, have been established as ubiquitous signal transduction components to control a diverse range of physiological pathways in higher plants (Bright et al., 2006; Tossi et al., 2012).The guard cell outward-rectifying K⁺ channel (GORK) encodes the exclusive voltage-gated outwardly rectifying K⁺ channel protein, which was located in the guard cell membrane (Ache et al., 2000; Dreyer and Blatt, 2009). Expression profiles revealed that this gene is up-regulated upon the onset of drought, salinity, and cold stress and ABA exposure (Becker et al., 2003; Tran et al., 2013). Reverse genetic evidence further showed that GORK plays an important role in the control of stomatal movements and allows the plant to reduce transpirational water loss significantly (Hosy et al., 2003) and participates in the regulation of salinity tolerance by preventing salt-induced K⁺ loss (Jayakannan et al., 2013). Due to the high complexity of guard cell signaling cascades, whether and how ABA-triggered GORK up-regulation is attributed to the generation of cellular secondary messengers, such as ROS and NO, is less clear.Hydrogen gas (H₂) was recently revealed as a signaling modulator with multiple biological functions in clinical trails (Ohsawa et al., 2007; Itoh et al., 2009; Ito et al., 2012). It was previously found that a hydrogenase system could generate H₂ in bacteria and green algae (Meyer, 2007; Esquível et al., 2011). Although some earlier studies discovered the evolution of H₂ in several higher plant species (Renwick et al., 1964; Torres et al., 1984), it was also proposed that the eukaryotic hydrogenase-like protein does not metabolize H₂ (Cavazza et al., 2008; Mondy et al., 2014). Since the explosion limit of H₂ gas is about 4% to 72.4% (v/v, in the air), the direct application of H₂ gas in experiments is flammable and dangerous. Regardless of these problems to be resolved, the methodology, such as using exogenous hydrogen-rich water (HRW) or hydrogen-rich saline, which is safe, economical, and easily available, provides a valuable approach to investigate the physiological function of H₂ in animal research and clinical trials. For example, hydrogen dissolved in Dulbecco’s modified Eagle’s medium was found to react with cytotoxic ROS and thus protect against oxidative damage in PC12 cells and rats (Ohsawa et al., 2007). The neuroprotective effect of H₂-loaded eye drops on retinal ischemia-reperfusion injury was also reported (Oharazawa et al., 2010). In plants, corresponding results by using HRW combined with gas chromatography (GC) revealed that H₂ could act as a novel beneficial gaseous molecule in plant responses against salinity (Xie et al., 2012; Xu et al., 2013), cadmium stress (Cui et al., 2013), and paraquat toxicity (Jin et al., 2013). More recently, the observation that HRW could delay the postharvest ripening and senescence of kiwifruit (Actinidia deliciosa) was reported (Hu et al., 2014).Considering the fact that the signaling cascades for salt, osmotic, and drought stresses share a common cascade in an ABA-dependent pathway, it would be noteworthy to identify whether and how H₂ regulates the bioactivity of ABA-induced downstream components and, thereafter, biological responses, including stomatal closure and drought tolerance. To resolve these scientific questions, rbohD, rbohF, nia1/2, nitric oxide associated1 (noa1; Van Ree et al., 2011), nia1/2/noa1, and gork mutants were utilized to investigate the relationship among H₂, ROS, NO, and GORK in the guard cell signal transduction network. By the combination of pharmacological and biochemical analyses with this genetics-based approach, we provide comprehensive evidence to show that H₂ might be a newly identified bioeffective modulator involved in ABA signaling responsible for drought tolerance, that HRW-promoted stomatal closure was mainly attributed to the modulation of ROS-dependent NO generation, and that GORK might be the downstream target protein of H₂ signaling.