Oxylipins and Reactive Carbonyls as Regulators of the Plant Redox and Reactive Oxygen Species Network under Stress.

Reactive oxygen species (ROS), and in particular H2O2, serve as essential second messengers at low concentrations. However, excessive ROS accumulation leads to severe and irreversible cell damage. Hence, control of ROS levels is needed, especially under non-optimal growth conditions caused by abiotic or biotic stresses, which at least initially stimulate ROS synthesis. A complex network of thiol-sensitive proteins is instrumental in realizing tight ROS control; this is called the redox regulatory network. It consists of sensors, input elements, transmitters, and targets. Recent evidence revealed that the interplay of the redox network and oxylipins-molecules derived from oxygenation of polyunsaturated fatty acids, especially under high ROS levels-plays a decisive role in coupling ROS generation and subsequent stress defense signaling pathways in plants. This review aims to provide a broad overview of the current knowledge on the interaction of distinct oxylipins generated enzymatically (12-OPDA, 4-HNE, phytoprostanes) or non-enzymatically (MDA, acrolein) and components of the redox network. Further, recent findings on the contribution of oxylipins to environmental acclimatization will be discussed using flooding, herbivory, and establishment of thermotolerance as prime examples of relevant biotic and abiotic stresses.

OPDAylation of Thiols of the Redox Regulatory Network In Vitro.

cis-(+)-12-Oxophytodienoic acid (OPDA) is a reactive oxylipin produced by catalytic oxygenation of polyunsaturated α-linolenic acid (18:3 (ω - 3)) in the chloroplast. Apart from its function as precursor for jasmonic acid synthesis, OPDA serves as a signaling molecule and regulator on its own, namely by tuning enzyme activities and altering expression of OPDA-responsive genes. A possible reaction mechanism is the covalent binding of OPDA to thiols via the addition to the C=C double bond of its α,ß-unsaturated carbonyl group in the cyclopentenone ring. The reactivity allows for covalent modification of accessible cysteinyl thiols in proteins. This work investigated the reaction of OPDA with selected chloroplast and cytosolic thioredoxins (TRX) and glutaredoxins (GRX) of Arabidopsis thaliana. OPDA reacted with TRX and GRX as detected by decreased m-PEG maleimide binding, consumption of OPDA, reduced ability for insulin reduction and inability to activate glyceraldehyde-3-phosphate dehydrogenase and regenerate glutathione peroxidase (GPXL8), and with lower efficiency, peroxiredoxin IIB (PRXIIB). OPDAylation of certain protein thiols occurs quickly and efficiently in vitro and is a potent post-translational modification in a stressful environment.

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s-1·mg protein-1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

α,ß-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,ß-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin-Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid.

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ2 and PGA2, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.

The redox-sensitive module of cyclophilin 20-3, 2-cysteine peroxiredoxin and cysteine synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response.

The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities is fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA), which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat2;1, cyp20-3 and 2-cysprxA/B, and wild-type (WT). Leaf transcriptomes were analyzed 6 h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared with WT. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism, and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling.

Redox-Dependent Conformational Dynamics of Decameric 2-Cysteine Peroxiredoxin and its Interaction with Cyclophilin 20-3.

2-Cysteine peroxiredoxins (2-CysPrxs) switch between functions as a thiol peroxidase, chaperone, an interaction partner and possibly a proximity-based oxidase in a redox-dependent manner. In photosynthetic eukaryotes, 2-CysPrx localizes to the plastid, functions in the context of photosynthesis and enables an ascorbate peroxidase-independent water-water cycle for detoxifying H2O2 The high degree of evolutionary conservation of 2-CysPrx suggests that the switching is an essential characteristic and needed to transduce redox information to downstream pathways and regulation. The study aimed at exploring the dissociation behavior of 2-CysPrx and its interactions with cyclophilin depending on bulk phase conditions. Isothermal titration microcalorimetry (ITC), dynamic light scattering and size exclusion chromatography (SEC) proved the previously suggested model that reduced 2-CysPrx below a critical transition concentration (CTC) exists in its dimeric state, and above the CTC adopts the decameric state. The presence of cyclophilin 20-3 (Cyp20-3) affected the CTC of a 2-CysPrx decamer suggesting interaction which was further quantified by direct titration of 2-CysPrx with Cyp20-3, and in overlays. Finally catalytic inactivation assays showed the higher catalytic efficiency of 2-CysPrx at pH 8 compared with pH 7.2, but also revealed increased inactivation by hyperoxidation at pH 8. Interestingly, calculation of the average turnover number until inactivation gave rather similar values of 243 and 268 catalytic cycles at pH 8 and pH 7.2, respectively. These quantitative data support a model where 2-CysPrx and Cyp20-3, by interaction, form a redox-sensitive regulatory module in the chloroplast which is under control of the photosynthesis-linked stromal pH value, the redox state and additional stromal protein factor(s).

Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana.

Plants are often simultaneously infested by several herbivores at the shoots and roots. Recent results revealed that the model plant Arabidopsis thaliana shows highly challenge-specific local and systemic responses to individual and simultaneous attacks of shoot-infesting aphids and root-infesting nematodes at the metabolome level. (1) Here, we present the corresponding transcriptional changes in plants treated with Brevicoryne brassicae aphids and Heterodera schachtii nematodes individually and in combination. Overall, shoots were much less responsive than roots. Gene expression in shoots and roots was mainly altered by aphids. Nematode infestation alone had only little effect, but nematodes modified the transcript accumulation response to aphids particularly in the roots. The responding genes are involved in plant defense cascades, signaling, oxidation-reduction processes, as well as primary and secondary metabolism and degradation. These changes in transcription may become relevant for the herbivores when they are translated into changes in host plant quality.

Alleviation of methyl viologen-mediated oxidative stress by Brassica juncea annexin-3 in transgenic Arabidopsis.

Plant annexins function as calcium-dependent or -independent phospholipid binding proteins and constitute about 0.1% of total cellular proteins. Some of them were reported to antagonize oxidative stress and protect plant cells. Brassica juncea annexin-3 (AnnBj3) was recently discovered. To gain insight into a possible function of AnnBj3 in oxidative stress response, we investigated the resistance of Arabidopsis thaliana plants expressing AnnBj3 constitutively. Here we report that, AnnBj3 attenuates methyl viologen-mediated oxidative stress in plants. It protected photosynthesis and plasma membrane from methyl viologen-mediated oxidative damage. AnnBj3 detoxifies hydrogen peroxide and showed antioxidative property in vitro. The protein increased total peroxidase activity in transgenics and interfered with other cellular antioxidants, thereby giving an overall cellular protection against methyl viologen-induced cytotoxicity.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae.

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

Efficient acclimation of the chloroplast antioxidant defence of Arabidopsis thaliana leaves in response to a 10- or 100-fold light increment and the possible involvement of retrograde signals.

Chloroplasts are equipped with a nuclear-encoded antioxidant defence system the components of which are usually expressed at high transcript and activity levels. To significantly challenge the chloroplast antioxidant system, Arabidopsis thaliana plants, acclimated to extremely low light slightly above the light compensation point or to normal growth chamber light, were moved to high light corresponding to a 100- and 10-fold light jump, for 6 h and 24 h in order to observe the responses of the water-water cycle at the transcript, protein, enzyme activity, and metabolite levels. The plants coped efficiently with the high light regime and the photoinhibition was fully reversible. Reactive oxygen species (ROS), glutathione and ascorbate levels as well as redox states, respectively, revealed no particular oxidative stress in low-light-acclimated plants transferred to 100-fold excess light. Strong regulation of the water-water cycle enzymes at the transcript level was only partly reflected at the protein and activity levels. In general, low light plants had higher stromal (sAPX) and thylakoid ascorbate peroxidase (tAPX), dehydroascorbate reductase (DHAR), and CuZn superoxide dismutase (CuZnSOD) protein contents than normal light-grown plants. Mutants defective in components relevant for retrograde signalling, namely stn7, ex1, tpt1, and a mutant expressing E .coli catalase in the chloroplast showed unaltered transcriptional responses of water-water cycle enzymes. These findings, together with the response of marker transcripts, indicate that abscisic acid is not involved and that the plastoquinone redox state and reactive oxygen species do not play a major role in regulating the transcriptional response at t=6 h, while other marker transcripts suggest a major role for reductive power, metabolites, and lipids as signals for the response of the water-water cycle.

AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling.

To optimize acclimation responses to environmental growth conditions, plants integrate and weigh a diversity of input signals. Signal integration within the signalling networks occurs at different sites including the level of transcription factor activation. Accumulating evidence assigns a major and diversified role in environmental signal integration to the family of APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factors. Presently, the Plant Transcription Factor Database 3.0 assigns 147 gene loci to this family in Arabidopsis thaliana, 200 in Populus trichocarpa and 163 in Oryza sativa subsp. japonica as compared to 13 to 14 in unicellular algae ( http://plntfdb.bio.uni-potsdam.de/v3.0/ ). AP2/EREBP transcription factors have been implicated in hormone, sugar and redox signalling in context of abiotic stresses such as cold and drought. This review exemplarily addresses present-day knowledge of selected AP2/EREBP with focus on a function in stress signal integration and retrograde signalling and defines AP2/EREBP-linked gene networks from transcriptional profiling-based graphical Gaussian models. The latter approach suggests highly interlinked functions of AP2/EREBPs in retrograde and stress signalling.