ABSTRACT
Reactive oxygen species (ROS) are critical factors that cause damage in salt-stressed plants, but their mechanisms of action in living cells are largely unknown. We investigated the roles of reactive carbonyl species (RCS), i.e., the lipid peroxide-derived α,ß-unsaturated aldehydes and ketones, in plant growth retardation under salt stress. When Arabidopsis thaliana Col-0 seeds were exposed to 100 mM NaCl, germination was delayed and the levels of ROS, RCS, and protein carbonylation in the seedlings were increased. Adding the histidine-containing dipeptides carnosine, N-acetylcarnosine, and anserine, which are reported RCS scavengers, restored the germination speed and suppressed the increases in RCS and protein carbonylation but did not affect the ROS level. Increases in the levels of the RCS acrolein, crotonaldehyde, (E)-2-pentenal, and 4-hydroxy-(E)-2-nonenal were positively correlated with the delay of germination and growth inhibition. These RCS, generated downstream of ROS, are thus primarily responsible for the salt-stress symptoms of plants.
Subject(s)
Arabidopsis , Histidine , Arabidopsis/metabolism , Dipeptides/metabolism , Histidine/metabolism , Plants/metabolism , Reactive Oxygen Species/metabolism , Salt StressABSTRACT
Reactive oxygen species (ROS) play a central role in plant responses to biotic and abiotic stresses. ROS stimulate stomatal closure by inhibiting blue light (BL)-dependent stomatal opening under diverse stresses in the daytime. However, the stomatal opening inhibition mechanism by ROS remains unclear. In this study, we aimed to examine the impact of reactive carbonyl species (RCS), lipid peroxidation products generated by ROS, on BL signaling in guard cells. Application of RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), inhibited BL-dependent stomatal opening in the epidermis of Arabidopsis thaliana. Acrolein also inhibited H+ pumping and the plasma membrane H+-ATPase phosphorylation in response to BL. However, acrolein did not inhibit BL-dependent autophosphorylation of phototropins and the phosphorylation of BLUE LIGHT SIGNALING1 (BLUS1). Similarly, acrolein affected neither the kinase activity of BLUS1 nor the phosphatase activity of protein phosphatase 1, a positive regulator of BL signaling. However, acrolein inhibited fusicoccin-dependent phosphorylation of H+-ATPase and stomatal opening. Furthermore, carnosine, an RCS scavenger, partially alleviated the abscisic-acid- and hydrogen-peroxide-induced inhibition of BL-dependent stomatal opening. Altogether, these findings suggest that RCS inhibit BL signaling, especially H+-ATPase activation, and play a key role in the crosstalk between BL and ROS signaling pathways in guard cells.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Acrolein/pharmacology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Membrane/metabolism , Light , Plant Stomata/physiology , Proton-Translocating ATPases/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Responses of plant cells to reactive oxygen species (ROS), e.g., reprogramming of defense genes or progression of cell death, should include the ROS signal transmission to target proteins, but the biochemistry of this process is largely unknown. Lipid peroxide-derived α,ß-unsaturated aldehydes and ketones (reactive carbonyl species; RCS), downstream products of ROS stimuli, are recently emerging endogenous agents that can mediate ROS signal to proteins via covalent modification. The involvement of RCS in certain ROS signaling in plants (oxidative injury of leaves and roots, ROS-induced programmed cell death, senescence, and abscisic acid and auxin signaling) has been verified by the determination of RCS with the use of conventional HPLC. Because distinct kinds of RCS act differently in the cell and so are metabolized, identification and quantification of each RCS in plant tissues provide central information to decipher biochemical mechanisms of plant responses to ROS. This article illustrates practical methods of plant sample preparation and extraction and analysis of RCS.
Subject(s)
Arabidopsis , Plant Cells , Abscisic Acid/metabolism , Arabidopsis/genetics , Oxidative Stress , Plant Cells/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Oxidation of membrane lipids by reactive oxygen species (ROS) or O2/lipoxygenase leads to the formation of various bioactive compounds collectively called oxylipins. Reactive carbonyl species (RCS) are a group of oxylipins that have the α,ß-unsaturated carbonyl structure, including acrolein and 4-hydroxy-(E)-2-nonenal. RCS provides a missing link between ROS stimuli and cellular responses in plants via their electrophilic modification of proteins. The physiological significance of RCS in plants has been established based on the observations that the RCS-scavenging enzymes that are overexpressed in plants or the RCS-scavenging chemicals added to plants suppress the plants' responses to ROS, i.e., photoinhibition, aluminum-induced root damage, programmed cell death (PCD), senescence, abscisic acid-induced stomata closure, and auxin-induced lateral root formation. The functions of RCS are thus a key to ROS- and redox-signaling in plants. The chemical species involved in distinct RCS signaling/damaging phenomena were recently revealed, based on comprehensive carbonyl determinations. This review presents an overview of the current status of research regarding RCS signaling functions in plants and discusses present challenges for gaining a more complete understanding of the signaling mechanisms.
ABSTRACT
The Arabidopsis thaliana aldehyde oxidase 3 (AAO3) catalyzes the oxidation of abscisic aldehyde (ABal) to abscisic acid (ABA). Besides ABal, plants generate other aldehydes that can be toxic above a certain threshold. AAO3 knockout mutants (aao3) exhibited earlier senescence but equivalent relative water content compared with wild-type (WT) during normal growth or upon application of UV-C irradiation. Aldehyde profiling in leaves of 24-day-old plants revealed higher accumulation of acrolein, crotonaldehyde, 3Z-hexenal, hexanal and acetaldehyde in aao3 mutants compared with WT leaves. Similarly, higher levels of acrolein, benzaldehyde, crotonaldehyde, propionaldehyde, trans-2-hexenal and acetaldehyde were accumulated in aao3 mutants upon UV-C irradiation. Aldehydes application to plants hastened profuse senescence symptoms and higher accumulation of aldehydes, such as acrolein, benzaldehyde and 4-hydroxy-2-nonenal, in aao3 mutant leaves as compared with WT. The senescence symptoms included greater decrease in chlorophyll content and increase in transcript expression of the early senescence marker genes, Senescence-Related-Gene1, Stay-Green-Protein2 as well as NAC-LIKE, ACTIVATED-BY AP3/P1. Notably, although aao3 had lower ABA content than WT, members of the ABA-responding genes SnRKs were expressed at similar levels in aao3 and WT. Moreover, the other ABA-deficient mutants [aba2 and 9-cis-poxycarotenoid dioxygenase3-2 (nced3-2), that has functional AAO3] exhibited similar aldehydes accumulation and chlorophyll content like WT under normal growth conditions or UV-C irradiation. These results indicate that the absence of AAO3 oxidation activity and not the lower ABA and its associated function is responsible for the earlier senescence symptoms in aao3 mutant.
Subject(s)
Abscisic Acid/metabolism , Aldehyde Oxidase/metabolism , Aldehydes/toxicity , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Plant Growth Regulators/metabolism , Aldehyde Oxidase/genetics , Aldehydes/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Chlorophyll/metabolism , Oxidation-Reduction , Plant Leaves/genetics , Plant Leaves/physiology , Plant SenescenceABSTRACT
Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of ß-carotene and, consequently, ß-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing ß-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from ß-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and ß-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from ß-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.
Subject(s)
Arabidopsis/metabolism , Carotenoids/metabolism , Plant Proteins/metabolism , Transcriptome , Xenobiotics/metabolism , Arabidopsis/genetics , Free Radicals/metabolism , Gene Expression Profiling , Plant Proteins/genetics , Plant Roots/genetics , Plant Roots/metabolism , Xanthophylls/metabolismABSTRACT
Lipid peroxidation-derived reactive carbonyl species (RCS) such as acrolein and 4-hydroxynonenal pose health risks. We characterized the RCS-scavenging reactions of tea catechins in an aqueous solution and in baked cake. Acrolein's reaction with each of the major tea catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) resulted in the formation of mono-, di-, and tri-acrolein conjugates of each catechin as revealed by our LC-linear ion trap MS analysis. The formation of the acrolein-conjugates of the four catechins was confirmed in the reaction of acrolein with green tea powder (matcha) extract. The addition of matcha tea powder to cake dough significantly suppressed the accumulation of RCS during cake baking. The mono-acrolein conjugates of the four major catechins were detected in the baked cake. The RCS-scavenging capability of tea catechins offers a new functionality of matcha tea powder, and its heat stability demonstrates the usefulness of matcha as a food additive.
Subject(s)
Acrolein/chemistry , Catechin/chemistry , Free Radical Scavengers/chemistry , Tea/chemistry , Acrolein/analysis , Aldehydes/chemistry , Catechin/analogs & derivatives , Catechin/analysis , Chromatography, High Pressure Liquid , Cooking , Hot Temperature , Mass Spectrometry , Plant Extracts/chemistry , Powders/chemistry , Tea/metabolismABSTRACT
Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ â 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.
Subject(s)
Acetates/metabolism , Cyclopentanes/metabolism , Oxylipins/metabolism , Plant Growth Regulators/physiology , Plant Stomata/physiology , Abscisic Acid/metabolism , Arabidopsis/metabolism , Arabidopsis/physiology , Hydrogen Peroxide/metabolism , Plant Growth Regulators/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Nicotiana/metabolism , Nicotiana/physiologyABSTRACT
The author wishes to make the following correction to this paper [...].
ABSTRACT
H2O2-induced programmed cell death (PCD) of tobacco Bright Yellow-2 (BY-2) cells is mediated by reactive carbonyl species (RCS), degradation products of lipid peroxides, which activate caspase-3-like protease (C3LP). Here, we investigated the mechanism of RCS accumulation in the H2O2-induced PCD of BY-2 cells. The following biochemical changes were observed in 10-min response to a lethal dose (1.0 mM) of H2O2, but they did not occur in a sublethal dose (0.5 mM) of H2O2. (1) The C3LP activity was increased twofold. (2) The intracellular levels of RCS, i.e., 4-hydroxy-(E)-hexenal and 4-hydroxy-(E)-nonenal (HNE), were increased 1.2-1.5-fold. (3) The activity of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent carbonyl reductase, scavenging HNE, and n-hexanal was decreased. Specifically, these are the earliest events leading to PCD. The proteasome inhibitor MG132 suppressed the H2O2-induced PCD, indicating that the C3LP activity of the 1 subunit of the 20S proteasome was responsible for PCD. The addition of H2O2 to cell-free protein extract inactivated the carbonyl reductase. Taken together, these results suggest a PCD-triggering mechanism in which H2O2 first inactivates a carbonyl reductase(s), allowing RCS levels to rise, and eventually leads to the activation of the C3LP activity of 20S proteasome. The carbonyl reductase thus acts as an ROS sensor for triggering PCD.
ABSTRACT
As reactive oxygen species (ROS) play critical roles in plants to determine cell fate in various physiological situations, there is keen interest in the biochemical processes of ROS signal transmission. Reactive carbonyl species (RCS), the ,-unsaturated aldehydes and ketones produced from lipid peroxides, due to their chemical property to covalently modify protein, can mediate ROS signals to proteins. Comprehensive carbonyl analysis in plants has revealed that more than a dozen different RCS, e.g., acrolein, 4-hydroxy-(E)-2-nonenal and malondialdehyde, are produced from various membranes, and some of them increase and modify proteins in response to oxidative stimuli. At early stages of response, specific subsets of proteins are selectively modified with RCS. The involvement of RCS in ROS signaling can be judged on three criteria: (1) A stimulus to increase the ROS level in plants leads to the enhancement of RCS levels. (2) Suppression of the increase of RCS by scavenging enzymes or chemicals diminishes the ROS-induced response. (3) Addition of RCS to plants evokes responses similar to those induced by ROS. On these criteria, the RCS action as damaging/signaling agents has been demonstrated for root injury, programmed cell death, senescence of siliques, stomata response to abscisic acid, and root response to auxin. RCS thus act as damage/signal mediators downstream of ROS in a variety of physiological situations. A current picture and perspectives of RCS research are presented in this article.
ABSTRACT
In auxin-stimulated roots, production of reactive oxygen species (ROS) via the hormone-induced activation of respiratory burst oxidase homologous NADPH oxidases facilitates lateral root (LR) formation. In this study, in order to verify that ROS can modulate auxin signaling, we examined the involvement of the lipid peroxide-derived agents known as reactive carbonyl species (RCS) in LR formation. When auxin was added to Arabidopsis thaliana roots, the levels of RCS, for example acrolein, 4-hydroxynonenal and crotonaldehyde, were increased prior to LR formation. Addition of the carbonyl scavenger carnosine suppressed auxin-induced LR formation. Addition of RCS to the roots induced the expression of the auxin-responsive DR5 promoter and the TIR1, IAA14, ARF7, LBD16 and PUCHI genes and facilitated LR formation without increasing the endogenous auxin level. DR5 and LBD16 were activated in the LR primordia. The auxin signaling-deficient mutants arf7 arf19 and slr-1 did not respond - and tir1 afb2 appeared to show a poor response - to RCS. When given to the roots RCS promoted the disappearance of the AXR3NT-GUS fusion protein, i.e. the degradation of the auxin/indole-3-acetic acid protein, as did auxin. These results indicate that the auxin-induced production of ROS and their downstream products RCS modulate the auxin signaling pathway in a feed-forward manner. RCS are key agents that connect the ROS signaling and the auxin signaling pathways.
Subject(s)
Arabidopsis/physiology , Free Radicals/metabolism , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Genes, Reporter , Lipid Peroxides/metabolism , Oxylipins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/physiology , Promoter Regions, Genetic/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Oxidative stimuli to living cells results in the formation of lipid peroxides, from which various aldehydes and ketones (oxylipin carbonyls) are inevitably produced. Among the oxylipin carbonyls, those with an α,ß-unsaturated bond are designated as reactive carbonyl species (RCS) because they have high electrophilicity and biological activity. Plants have arrays of dehydrogenases and reductases to metabolize a variety of RCS that occur in the cells, but these enzymes are not efficient to scavenge the most toxic RCS (i.e., acrolein) because they have only low affinity. Two glutathione transferase (GST) isozymes belonging to the plant-specific Tau class were recently observed to scavenge acrolein with K M values at a submillimolar level. This suggests that GST could also be involved in the defense system against RCS. We tested the activities of 23 Tau isozymes of Arabidopsis thaliana for five types of RCS, and the results revealed that 11 isozymes recognized either acrolein or 4-hydroxy-(E)-2-nonenal or both as a substrate(s). Such RCS-scavenging activities indicate the potential contribution of GST to RCS scavenging in plants, and they may account for the stress tolerance conferred by several Tau isozymes. RCS are therefore a strong candidate for endogenous substrates of plant GSTs.
ABSTRACT
We have demonstrated that reactive carbonyl species (RCS) function as an intermediate downstream of hydrogen peroxide (H2O2) production in abscisic acid (ABA) signaling for stomatal closure in guard cells using transgenic tobacco plants overexpressing alkenal reductase. We investigated the conversion of the RCS production into downstream signaling events in the guard cells. Both ABA and H2O2 induced production of the RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), in epidermal tissues of wild-type Arabidopsis thaliana plants. Application of the RCS scavengers, carnosine and pyridoxamine, did not affect the ABA-induced H2O2 production but inhibited the ABA- and H2O2-induced stomatal closure. Both acrolein and HNE induced stomatal closure in a plasma membrane NAD(P)H oxidase mutant atrbohD atrbohF as well as in the wild type, but not in a calcium-dependent kinase mutant cpk6. Acrolein activated plasma membrane Ca2+-permeable cation channels, triggered cytosolic free Ca2+ concentration ([Ca2+]cyt) elevation, and induced stomatal closure accompanied by depletion of glutathione in the guard cells. These results suggest that RCS production is a signaling event between the ROS production and [Ca2+]cyt elevation during guard cell ABA signaling.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Hydrogen Peroxide/metabolism , Phytochrome/metabolism , Signal TransductionABSTRACT
Oxidation of membrane lipids by reactive oxygen species primarily generates lipid peroxides, from which various carbonyls, i.e., aldehydes and ketones, are formed. Among them, those with a carbonyl-conjugated C-C double bond have significant biological functions and are designated as reactive carbonyl species (RCS). A dozen kinds of RCS occurring in plant cells have a broad spectrum of reactivity and biological effects, depending on the structure. Several RCS have been recently found to activate caspase-like proteases in plants, thereby initiating PCD. Comprehensive and quantitative RCS analysis method using conventional HPLC is illustrated.
Subject(s)
Oxidation-Reduction , Oxidative Stress , Plants/metabolism , Reactive Oxygen Species/metabolism , Chromatography, High Pressure Liquid , Hydrazines/metabolism , Plant Physiological PhenomenaABSTRACT
Lipid peroxide-derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage-inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR-OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild-type under Al stress. Exogenous application of 4-hydroxy-2-nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild-type plants. GR-OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H2 O2 due to Al stress in GR-OE plants was lower by 26% than in wild-type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)-2-heptenal and n-octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.
Subject(s)
Aluminum/toxicity , Arabidopsis Proteins/metabolism , Arabidopsis/drug effects , Glutathione Reductase/metabolism , Glutathione/metabolism , Reactive Oxygen Species/metabolism , Aldehydes/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Ascorbic Acid/metabolism , Glutathione Reductase/genetics , Hydrogen Peroxide/metabolism , Plant Roots/drug effects , Plant Roots/growth & development , Plants, Genetically Modified , Sulfhydryl Compounds/metabolismABSTRACT
MAIN CONCLUSION: Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K M isozyme from spinach. Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K M value for acrolein 93 µM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.
Subject(s)
Acrolein/metabolism , Glutathione Transferase/metabolism , Plant Proteins/metabolism , Spinacia oleracea/enzymology , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Chromatography, Affinity , Chromatography, Ion Exchange , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Inactivation, Metabolic , Isoenzymes/genetics , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Plant Leaves/enzymology , Plant Proteins/isolation & purification , Sequence Homology, Amino AcidABSTRACT
Drought is responsible for a massive reduction in crop yields. In response to drought, plants synthesize the hormone ABA, which induces stomatal closure, thus reducing water loss. In guard cells, ABA triggers production of reactive oxygen species (ROS), which is mediated by NAD(P)H oxidases. The production of ROS is a key factor for ABA-induced stomatal closure, but it remains to be clarified how the production of ROS is transduced into downstream signaling components in guard cells. We investigated roles of reactive carbonyl species (RCS) in ABA-induced stomatal closure using transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis 2-alkenal reductase (AER-OE), which scavenges RCS. ABA and hydrogen peroxide (H2O2) induced accumulation of RCS including acrolein and 4-hydroxy-(E)-2-nonenal in wild-type tobacco but not in AER-OE. Stomatal closure and RCS accumulation in response to ABA and H2O2 were inhibited in AER-OE unlike in the wild type, while ABA-induced H2O2 production in guard cells was observed in AER-OE as well as in the wild type. Moreover, ABA inhibited inward-rectifying K+ channels in wild-type guard cells but not in AER-OE guard cells. These results suggest that RCS is involved in ABA-induced stomatal closure and functions downstream of H2O2 production in the ABA signaling pathway in guard cells.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis/genetics , Free Radicals/metabolism , Nicotiana/physiology , Plant Growth Regulators/metabolism , Signal Transduction , Arabidopsis Proteins/genetics , Droughts , Free Radicals/analysis , Gene Expression , Hydrogen Peroxide/analysis , Hydrogen Peroxide/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Plant Stomata/genetics , Plant Stomata/physiology , Plants, Genetically Modified , Reactive Oxygen Species/analysis , Reactive Oxygen Species/metabolism , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Reactive oxygen species (ROS)-triggered programmed cell death (PCD) is a typical plant response to biotic and abiotic stressors. We have recently shown that lipid peroxide-derived reactive carbonyl species (RCS), downstream products of ROS, mediate oxidative signal to initiate PCD. Here we investigated the mechanism by which RCS initiate PCD. Tobacco Bright Yellow-2 cultured cells were treated with acrolein, one of the most potent RCS. Acrolein at 0.2 mM caused PCD in 5 h (i.e. lethal), but at 0.1 mM it did not (sublethal). Specifically, these two doses caused critically different effects on the cells. Both lethal and sublethal doses of acrolein exhausted the cellular glutathione pool in 30 min, while the lethal dose only caused a significant ascorbate decrease and ROS increase in 1-2 h. Prior to such redox changes, we found that acrolein caused significant increases in the activities of caspase-1-like protease (C1LP) and caspase-3-like protease (C3LP), the proteases which trigger PCD. The lethal dose of acrolein increased the C3LP activity 2-fold more than did the sublethal dose. In contrast, C1LP activity increments caused by the two doses were not different. Acrolein and 4-hydroxy-(E)-2-nonenal, another RCS, activated both proteases in a cell-free extract from untreated cells. H2O2 at 1 mM added to the cells increased C1LP and C3LP activities and caused PCD, and the RCS scavenger carnosine suppressed their activation and PCD. However, H2O2 did not activate the proteases in a cell-free extract. Thus the activation of caspase-like proteases, particularly C3LP, by RCS is an initial biochemical event in oxidative signal-stimulated PCD in plants.