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.     

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.     

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.     

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton

Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.Cotton (Gossypium spp.) is one of the most economically important crops worldwide and a number of pathogens affect the growth and development of cotton plants. The soil-borne pathogen Verticillium dahliae (V. dahliae) causes the destructive vascular disease Verticillium wilt, which results in devastating reductions in plant mass, lint yield, and fiber quality (Bolek et al., 2005; Cai et al., 2009). To date, Verticillium wilt has not been effectively controlled in the most common cultivated cotton species, upland cotton (Gossypium hirsutum), and cultivars with stably inherited resistance to this disease are currently unavailable (Aguado et al., 2008; Jiang et al., 2009; Zhang et al., 2012a). Unlike upland cotton, sea-island cotton (Gossypium barbadense), which is only cultivated on a small scale, possesses Verticillium wilt resistance. Exploring the molecular mechanisms involved in the defense responses against V. dahliae invasion in G. barbadense can provide useful information for generating wilt-resistant G. hirsutum species through molecular breeding.During the past decades, progress has been made in studying the defense responses against V. dahliae infection in cotton. Global analyses have demonstrated that several signaling pathways, including those mediated by salicylic acid, ethylene, jasmonic acid, and brassinosteroids, activate distinct processes involved in V. dahliae defense (Bari and Jones, 2009; Grant and Jones, 2009; Gao et al., 2013a). Accumulating evidence indicates that many V. dahliae-responsive genes, such as GbWARKY1, GhSSN, GbERF, GhMLP28, GhNDR1, GhMKK2, and GhBAK1 (Qin et al., 2004; Gao et al., 2011, 2013b; Li et al., 2014a; Sun et al., 2014; Yang et al., 2015), play crucial roles in defense against Verticillium wilt. In addition, the biosynthesis of terpenoids, lignin, and gossypol also makes important contributions to V. dahliae resistance in cotton (Tan et al., 2000; Luo et al., 2001; Xu et al., 2011; Gao et al., 2013a). Together, these studies have greatly improved our understanding of the complex innate defense systems against V. dahliae infection in cotton.The initial interaction between plants and pathogens takes place in the apoplast, the compartment of the plant cell outside the cell membrane, including the cell wall and intercellular space (Dietz, 1997). In response to pathogen colonization, the attacked plant cells undergo significant cellular and molecular changes, such as reinforcement of the cell wall and secretion of antimicrobial molecules into the apoplastic space (Bednarek et al., 2010). Thus, the apoplast serves as the first line of defense against microbe invasion, and apoplast immunity can be considered an important component of the plant immune response to pathogens.Upon recognition of pathogen infection, rapid production of reactive oxygen species [the reactive oxygen species (ROS) burst] occurs in the apoplast (Lamb and Dixon, 1997; Torres et al., 2006; Torres, 2010). This ROS burst is regarded as a core component of the early plant immune response (Daudi et al., 2012; Doehlemann and Hemetsberger, 2013). During defense responses, apoplastic ROS can diffuse into the cytoplasm and serve as signals, interacting with other signaling processes such as phosphorylation cascades, calcium signaling, and hormone-mediated pathways (Kovtun et al., 2000; Mou et al., 2003). Apoplastic ROS can also directly strengthen the host cell walls by oxidative cross linking of glycoproteins (Bradley et al., 1992; Lamb and Dixon, 1997) or the precursors of lignin and suberin polymers (Hückelhoven, 2007). Moreover, apoplastic ROS can directly affect pathogens by degrading nucleic acids and peptides from microbes or causing lipid peroxidation and membrane damage in the microbe (Mehdy, 1994; Lamb and Dixon, 1997; Apel and Hirt, 2004; Montillet et al., 2005).ROS levels in the apoplast increase rapidly in response to a variety of pathogens, but subsequently return to basal levels. The rapid production and dissipation of apoplastic ROS indicate that this process is finely regulated. Two classes of enzymes, NADPH oxidases and class III peroxidases, account for the rapid ROS burst in the apoplast (Bolwell et al., 1995; O’Brien et al., 2012). NADPH oxidases are directly phosphorylated by the receptor-like kinase BIK1 to enhance ROS generation (Li et al., 2014b). Also, due to the toxicity of high levels of ROS, plants have evolved enzymatic and nonenzymatic mechanisms to eliminate ROS, thereby preventing or reducing oxidative damage (Rahal et al., 2014; Torres et al., 2006). However, the molecular system responsible for the regulation of apoplastic ROS homeostasis during the immune response is not well understood.In this study, we performed a comparative analysis of the apoplastic proteomes in control roots compared with V. dahliae-inoculated roots of Gossypium barbadense (wilt-resistant sea-island cotton) using the two-dimensional differential gel electrophoresis (2D-DIGE) technique. Among the differentially expressed apoplastic proteins, ROS-related proteins were found to be major components, including a thioredoxin, GbNRX1, which functions as an ROS scavenger in response to V. dahliae infection. Knock-down of GbNRX1 expression in cotton by virus-induced gene silencing (VIGS) resulted in reduced resistance to V. dahliae. Our results demonstrate that maintaining apoplastic ROS homeostasis is a crucial component of the apoplastic immune response and that GbNRX1 is an important regulator of this process.     

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.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis

Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS), necessitating a redox stress response. However, little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here, we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKα) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD), which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKα leads to reduced G6PD activity, elevated levels of ROS, and enhanced sensitivity to salt stress. Conversely, plants overexpressing ASKα have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKα stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467, which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response.     

Loss of Ceramide Kinase in Arabidopsis Impairs Defenses and Promotes Ceramide Accumulation and Mitochondrial H2O2 Bursts

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.     

A Mediator of Singlet Oxygen Responses in Chlamydomonas reinhardtii and Arabidopsis Identified by a Luciferase-Based Genetic Screen in Algal Cells

All cells produce reactive oxygen species (ROS) as by-products of their metabolism. In addition to being cytotoxic, ROS act as regulators of a wide range of developmental and physiological processes. Little is known about the molecular mechanisms underlying the perception of ROS and initiation of cellular responses in eukaryotes. Using the unicellular green alga Chlamydomonas reinhardtii, we developed a genetic screen for early components of singlet oxygen signaling. Here, we report the identification of a small zinc finger protein, METHYLENE BLUE SENSITIVITY (MBS), that is required for induction of singlet oxygen–dependent gene expression and, upon oxidative stress, accumulates in distinct granules in the cytosol. Loss-of-function mbs mutants produce singlet oxygen but are unable to fully respond to it at the level of gene expression. Knockout or knockdown of the homologous genes in the higher plant model Arabidopsis thaliana results in mutants that are hypersensitive to photooxidative stress, whereas overexpression produces plants with elevated stress tolerance. Together, our data indicate an important and evolutionarily conserved role of the MBS protein in ROS signaling and provide a strategy for engineering stress-tolerant plants.     

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.