POG PP 2.1:
Potassium nitrate and hydrogen peroxide as seed germination promoters: modulation of antioxidant metabolism and hormone profile
Dr. José Antonio Hernández Cortés | CEBAS-CSIC | Spain
Seed germination is the most critical stage in crop establishment, determining crop production. Seed chemical treatment during imbibition has been successfully applied for both fundamental research purposes and the stimulation of seed germination, seedling vigour and seed dormancy alleviation . In the present work, we analysed the effect of H2O2 and of KNO3 treatments during imbibition in the germination and seedling growth of peach and pea seeds, respectively. With regard to peach seeds, both whole and endocarp-less seeds were treated with 10 mM H2O2 following cold stratification. For pea seeds, imbibition in KNO3 was applied at concentrations from 0.25 to 80 mM KNO3.
We observed that a low KNO3 level led to enhanced seedling water uptake and growth, increased activity of some antioxidant enzymes, and decreased levels of reduced ascorbate and glutathione. In addition, the levels of gibberellin GA1 increased whereas abscisic acid (ABA) contents decreased both in the seedlings and in the cotyledons, resulting in a decline in the ABA/GAs ratio .
On the other hand, H2O2 imbibition of endocarp-less peach seeds following 8 weeks of stratification, increased germination rate and seedling vigour. The H2O2 imbibition also affected the levels of non-enzymatic antioxidants ascorbate and glutathione, as well as ABA and jasmonic acid content in peach seedlings .
Overall, KNO3 and H2O2 were proven to be efficient seed germination promoters, modulating antioxidant defences and hormone profile, which was also reflected in improved seedling growth.
 Barba-Espin et al. (2010) Plant Cell Environ., 33, 981–994.
 Hernández et al (2022). Seeds, 1, 5-15
 Barba-Espín et al (2022). Seeds, 1, 28-35
Funded by the project DEVELOPMENT AND OPTIMIZATION OF AGRICULTURAL SYSTEMS BASED ON HALOPHITES IN MEDITERRANEAN SOILS AFFECTED BY SALT (PCI2020-111977), MCIN/AEI/10.13039/501100011033 and by the European Union “NextGenerationEU”/PRTR”, PRIMA Program (PRIMA S2 2019); MCIN; AEI 10.13039/501100011033 and PRTR.
POG PP 2.2:
Arabidopsis class I glutaredoxins in the cytosol and endoplasmic reticulum have different oxidoreductase activities
Dr Anna Moseler | University of Bonn | Germany
Glutaredoxins (GRXs) constitute a subfamily of the thioredoxin (TRX) superfamily of oxidoreductases. Although they are characterized by a conserved core structure, the so-called TRX-fold, they show different catalytic activities and functions. In Arabidopsis, GRXs form a large family with 31 members and several subclasses. The first class comprises five GRXs termed GRXC1-5 with a CxxC active site motif plus a close isoform of GRXC5, GRXS12 with a CxxS motif. While GRXC5 and GRXS12 are localized in plastids and have been described earlier, the localization and function of the other class I members is less clear. Thus, our aim is to analyze the precise subcellular localization of GRXC1-GRXC4 by using the redox-sensitive GFP2 (roGFP2) as visible tag. In addition, the function of these GRXs is investigated by combining biochemical activity assays with genetics and characterization of null mutants. Stable expression of the roGFP2 fusion proteins in Arabidopsis show a cytosolic localization for GRXC1 and GRXC2 with GRXC1 being attached to membranes through N-terminal myristoylation. Furthermore, we reveal that GRXC3 and GRXC4 are type II membrane proteins with the catalytic domains facing the lumen of the endomembrane system. In accordance to their different localization sites, we found distinct oxidoreductase activities for GRXC1/2 on the one hand and GRXC3/4 on the other. Taking the advantage of roGFP2 as a biosensor that relies of catalytic activity of GRXs for oxidation and reduction, we show that GRXC1/2 have an enhanced capacity to reduce roGFP2, while GRXC3/4 are more efficiently oxidizing roGFP2. In Arabidopsis, the analysis of the respective single mutants and the grxc1 grxc2 or grxc3 grxc4 double mutant does not display any pronounced phenotype under normal growth conditions. We now seek to further elucidate the cause of the different catalytic properties of the GRXs and if they are required under certain stress conditions.
POG PP 2.3:
Identification of intramembrane proteases that activate membrane-bound transcription factors during mitochondrial retrograde regulation
Jonas De Backer | Ghent University; VIB Center for Plant Systems Biology | Belgium
Due to their sessile lifestyle, plants are exposed to ever-changing and often stressful environments, such as drought, heat, and pathogen assaults. To survive these harmful conditions, plants evolved to have complex mechanisms to recognize and counteract these conditions. Besides the plasma membrane, intracellular organelles such as chloroplasts and mitochondria are in a prime position for sensing and reporting stress signals to the nucleus to regulate stress-responsive gene expression. The molecular mechanisms of these organelle-to-nucleus (also referred to as retrograde) signaling networks are not well understood in plants. Our lab identified a novel mitochondrial retrograde signaling pathway, in which transcription factors of the NO APICAL MERISTEM/ ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/ CUPSHAPED COTYLEDON (NAC) family that are anchored to the ER -membranes through their C-terminal transmembrane domain play a key role. Upon mitochondrial perturbation by stresses, the N-terminal part of these transcription factors is released from the ER-membranes and translocated to the nucleus to regulate stress-responsive gene expression. However, the molecular mechanisms that underlie the release of these transcription factors and how mitochondria signal to the ER during plant stress responses remain not well understood. In silico and pharmacological analysis indicate that these ER-anchored NAC transcription factors are cleaved by rhomboid proteases. The aim of my PhD project is to identify the responsible proteases by activity-based-protein profiling and proximity-based labeling approaches and consequently unravel the molecular mechanisms of ER-membrane bound transcription factor activation during mitochondrial retrograde signaling of plant stress responses.
POG PP 2.4:
Transcriptome and proteome mining of H2S metabolism at the subcellular level of sweet pepper (Capsicum annuum L.) fruits during ripening and under a nitric oxide (NO)-enriched environment
Dr. Salvador González-Gordo | Estación Experimental del Zaidín (Spanish National Research Council, CSIC) | Spain
Omics technologies have become powerful tools to get deeper insights into the mechanism of regulation of the different metabolic pathways. Fruit ripening is a natural physiological process that involves drastic modifications at gene, protein, and metabolite levels. Using the transcriptome and proteome of sweet pepper (Capsicum annuum L.) fruits obtained by RNAseq and iTRAQ approaches, respectively, this study focuses on the identification and modulation of the genes and proteins involved in the metabolism of hydrogen sulfide (H2S) in the main subcellular compartments including cytosol, plastids, and mitochondria at different ripening stages: immature green, ripe red, and fruits subjected to nitric oxide (NO) gas treatment. The obtained data allowed the identification of 26 components distributed in the different subcellular compartments being differentially modulated during ripening and by NO. To our knowledge, this analysis provides the first framework for the metabolism of H2S in this non-climacteric fruit.
[Supported by a European Regional Development Fund cofinanced grants from the Ministry of Science and Innovation (PID2019-103924GB-I00) and Junta de Andalucía (P18-FR-1359), Spain]
POG PP 2.5:
Elevated CO2 mitigates the impact of drought stress and oxidative stress by upregulating glucosinolate metabolism in Arabidopsis thaliana
Prof. Han Asard | University of Antwerp | Belgium
Elevated CO2 (eCO2) reduces the impact of drought, but the mechanisms underlying this effect remain unclear. We used a multidisciplinary approach to investigate the effect of drought and eCO2 (620 ppm) on growth and oxidative stress of Arabidopsis thaliana leaves. Transcriptome and subsequent metabolite analyses identified a strong induction of the glucosinolate (GL) biosynthesis as a main effect of eCO2 in drought- stressed leaves. Of the 53 genes involved in GL metabolism and regulation, 26, mainly involved in aliphatic GL metabolism, were differentially expressed in response to eCO2 and/or drought. Transcriptome results highlighted the upregulation of ABI5 and downregulation of WRKY63 transcription factors (TF), known to enhance and inhibit the expression of genes regulating the aliphatic GL biosynthesis (e.g., MYB28 and MYB29 TFs, respectively. In addition, eCO2 positively regulated aliphatic GL biosynthesis by bHLH-mediated signalling via MYB TF and increasing the accumulation of GL precursors, in particular methionine. To prove the role of GL metabolism in the stress mitigating impact of eCO2, we exposed two mutants, deficient in aliphatic GLs (cyp79f2 and cyp79f1f2), and three aliphatic GL overexpressing transgenic lines (35S:MYB76, 35S:MYB29 and 35S:MYB28), to drought and eCO2. Overexpression of MYB TFs improved drought tolerance by inducing stomatal closure and maintaining plant turgor, whereas loss of cyp79f genes reduced the stress mitigating effect of eCO2 and decreased drought tolerance. Thus, we conclude that the GL metabolism plays a role in the stress mitigating effect of eCO2.
POG PP 2.6:
Phytoglobin expression alters the Na+/K+ balance and antioxidant responses in soybean plants exposed to Na2SO4
Claudio Stasolla | University of Manitoba | Canada
Soybean (Glycine max) is an economically important crop which is very susceptible to salt stress. Tolerance to Na2SO4 stress was evaluated in soybean plants over-expressing or suppressing the phytoglobin GmPgb1. Salt stress depressed several gas exchange parameters, including photosynthetic rate, caused leaf damage, and reduced water content and dry weights. Lower expression of Respiratory Burst Oxidase Homologs (RBOHB and D), as well as enhanced antioxidant activity, resulting from GmPgb1 overexpression, limited ROS-induced damage in salt-stressed leaf tissue. The leaves also exhibited higher activities of the H2O2-quenching enzymes, catalase (CAT) and ascorbate peroxidase (APX), as well as enhanced levels of ascorbic acid. Relative to WT and GmPgb1-suppressing plants, over-expression of GmPgb1 attenuated the accumulation of foliar Na+ and exhibited a lower Na+/K+ ratio. These changes were attributed to the induction of the Na+ efflux transporter SALT OVERLY SENSITIVE 1 (SOS1) limiting Na+ intake and transport, and the inward rectifying K+ channel, POTASSIUM TRANSPORTER 1 (AKT1), required for the maintenance of the Na+/K+ balance.
POG PP 2.7:
Shifts in redox balance with senescence trigger NO decay in Arabidopsis WT plants
Prof. Dr. Ewa Sobieszczuk-Nowicka | Adam Mickiewicz University in Poznań, Poland, Department of Plant Physiology | Poland
To determine if the cellular redox status created by the developmental phase affects nitric oxide (NO) level, we monitored this signal molecule during 7-day-dark-induced leaf senescence (DILS). DILS was used as a model for the perturbation of redox homeostasis and overproduction of reactive oxygen species. Senescence promoted an unfavorable oxidative environment for NO production. Measuring redox parameters including glutathione couple (GSH/GSSG ratio) we found that the senescence-like phenomenon was effective in cellular homeostasis imbalance, and provoked accumulation of hydrogen peroxide starting from day 3 of DILS. Electrodetection in extracts of individually darkened WT Arabidopsis leaves (leaf 7 of the rosette) revealed a significant drop in NO signal from day 1 to day 7. Notably, reversing the DILS program by restoring light access on day 3 recovered the NO pool, increased chlorophyll content, and enhanced GSH/GSSG ratio. Taken together, significant shift in redox balance favoring decrease in NO emission is an important element in reprogramming the overall organization of senescing events.
This work was supported by Polish National Science Centre in the frame of the project UMO-2017/26/E/NZ4/00226 (MAJ)
POG PP 2.8:
Identification of nitrotryptophan-containing proteins in Arabidopsis WT leaves undergoing dark-induced senescence
Dr Przemysław Jagodzik | Department of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University | Poland
Transfer of nitric oxide message into biological function includes interactions with specific residues of target proteins that undergo NO-dependent posttranslational modifications (PTM) among which S-nitrosation and tyrosine (Tyr) nitration are the most widely recognized. Unlike protein Tyr nitration, there is no information available on protein tryptophan (Trp) nitration in plant senescence or senescence-like phenomena. To identify the potential in vivo targets of Trp nitration in Arabidopsis WT leaves undergoing dark-induced senescence (DILS), a protein immunoprecipitation (IP) with an anti-6-nitroTrp antibody was performed. Day 3 of DILS was selected as the time point corresponding with the most abundant profile of 6-nitroTrp containing proteins. Mass spectrometry analysis of IP eluates revealed 64 characteristic proteins for control leaves (such as dihydrolipoyldehydrogenase 1, 60S ribosomal protein L13a-1, glutamine synthetase cytosolic isozyme 1-3). As many as 6 proteins were detected in both control and undergoing dark-induced senescence leaves. These included: S-adenosylmethionine synthase 2, malate dehydrogenase 1, bifunctional L-3-cyanoalanine synthase/cysteine synthase C1, GF14 protein phi chain, protein EXORDIUM-like 4 and plastid-lipid-associated protein 6. Four proteins were described as exclusively characteristic for DILS - beta-D-xylosidase 1, glyoxylate/succinic semialdehyde reductase 1, dihydrolipoamide dehydrogenase 1 and probable voltage-gated potassium channel subunit beta. The categories containing the highest number of identified protein candidates for Trp nitration in Arabidopsis leaves were related to protein biosynthesis and carbohydrate metabolism. Taken together, the results present the first catalogue of nitroTrp-containing proteins and indicate protein Trp-nitration as a selective and regulatory PTM during optimal development that declines during senescence-like phenomena.
This work was supported by Polish National Science Centre in the frame of the project UMO-2017/26/E/NZ4/00226
POG PP 2.9:
Role of Nitric Oxide Synthases from Klebsormidium nitens: first structural characterization and partners identification
Pauline Chatelain | Inrae - UMR Agroecologie | France
Nitric oxide (NO) is an important cellular signaling molecule regulating various physiological processes, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have been measured, although no sequences encoding mammalian NOSs have been found in land plants. Interestingly, we identified NOS-like sequences in 20 algae species. These latter include the filamentous charophyte green algae Klebsormidium nitens, a biological model to study the early transition step from aquatic algae to land plants.
In order to understand the mechanisms governing NO synthesis and signaling in green lineage we initiated the functional characterization of K. nitens NOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response to abiotic stresses. Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possesses classical mammalian NOS architecture consisting of oxygenase and reductase domains with some specificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2 displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seem to be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditions tested, whereas KnNOS2 appeared to be transcriptionally regulated during stress.
In parallel studies, we also built the in silico protein–protein interaction network of human NOSs using the BioGRID database and human NOS interaction data. Interestingly, genes encoding orthologs of several of these candidates were found in K. nitens genome. Some of these conserved partners are known to be involved in mammalian NOSs regulation and represent interesting candidates for further investigation.
Overall these findings open the way for a deeper characterization of KnNOSs and its protein partners and will facilitate further investigation of NO signaling in green lineage.
POG PP 2.10:
An Exploration of Mitochondrial Respiration and ROS in the Green Alga Chlamydomonas reinhardtii
Mitchell Ticoras | The Ohio State University | United States
In this study, we examined the cellular response to ROS-associated oxidative stress in instances of mitochondrial respiratory dysfunction. Our model of study is the green alga Chlamydomonas reinhardtii. As a preliminary assessment of the cellular ROS response, we monitored the growth of a collection of algal respiratory deficient mutants via tenfold dilution series. The cells were challenged with exogenously applied H2O2 and different light intensities that we presume augment internal ROS production. From this study, we uncovered that complex I (CI) mutants, regardless of their degree of defect in holoenzyme assembly and/or activity, do not appear to be more resistant or sensitive when exposed to our ROS treatment. This suggests that mutants impaired for CI function do not produce deleterious amounts of ROS unlike what has been reported in the context of mitochondrial CI dysfunction in humans. One possible explanation that accounts for this discrepancy is the operation of the natural CI bypass in plants, namely alternative NADH dehydrogenases, which are nonexistent in human cells. Interestingly, complex III (CIII) null mutants displayed an enhanced sensitivity when challenged with ROS. CIII mutants that have partially reverted to respiratory proficiency, however, display wildtype-like resistance to ROS treatment. This suggests that CIII deficiency may result in increased internal ROS production within the cell that causes death when additional external oxidative stress is applied. Alternatively, CIII function could be necessary to activate the physiological response necessary to detoxify ROS as evidenced in another model system, the yeast Saccharomyces cerevisiae. Moving forward, we intend to assay the expression and activity of ROS-detoxifying machinery in CIII deficient and wild-type cells.
POG PP 2.11:
Optimizing sulfenylated peptide identification of labelled proteins in the green microalga Chlamydomonas reinhardtii
Anna Caccamo | Université de Liège | Belgium
In photosynthetic organisms, chloroplast is one of the main sources of reactive oxygen species (ROS). The most common ROS produced during photosynthesis are superoxide anion (O2-), singlet oxygen (1O2) and hydrogen peroxide (H2O2) . In this study we mainly focused on H2O2 which can also act as a signalling molecule by oxidizing cysteine residues of proteins into their sulfenylated form. The aim of the project is to identify these proteins in order to establish their role during oxidative stress in the green microalga C. reinhardtii. We optimized a protocol for trapping sulfenylated proteins from  by using a benzothiazine-based chemoselective probe (BTD)  specific to recognize -SOH. We succeeded to label sulfenylated proteins after addition of H2O2. However, we are still in the process to optimize the workflow for modified peptides identification by mass spectrometry analyses. Preliminary results on wild type cells have shown that some photosynthetic proteins are susceptible to H2O2 modification, such us the light-harvesting complexes (LHC) of photosysthem II, LHCII3, LHCB3 and LHCII-1.3 important to capture light during photosynthesis. Our analyses will provide information on the Chlamydomonas sulfenome, and will be compared to the other post translational modifications already identified on the Chlamydomonas proteome, such as glutathionylation and nitrosylation.
Funded by FNRS-FWO EOS Project 30829584
 Asada (2006). Plant Physiol. 141 (2): 391-396
 Huang et al. (2019). PNAS. 116 (42): 21256-21261
 Gupta et al. (2017) J. Am. Chem. Soc. 139 (159): 5588–5595
POG PP 2.12:
Unraveling the interplay between Cu and drought combined action in barley plants – a double trouble?
Maria Martins | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
The indiscriminate use of copper (Cu) in agriculture results in its accumulation in soils and consequent phytotoxicity, being this problem aggravated by the increasing frequency and intensity of soil drought events. Since the impact of the interaction of these stressors on plant physiology remains unexplored, this study aimed to evaluate the combined action of Cu and drought on barley (Hordeum vulgare L.) plants. Following a bifactorial design, seedlings were grown in a natural soil for 14 d under the following treatments: a) control (CTL) – plants continuously irrigated (14 d) in an uncontaminated soil; b) Cu – plants continuously irrigated (14 d) in a Cu-contaminated soil (115 mg Cu kg-1); c) drought – plants only irrigated during the first 7 d of growth in an uncontaminated soil; d) combined – plants co-exposed to Cu and drought treatments. Results showed that while root length was negatively affected by Cu (individually and combined), drought stress (individually and combined) led to a decreased plant biomass, compared to the CTL. Regarding ROS production, superoxide anion levels significantly decreased in leaves of Cu treatments and increased in roots of plants exposed to the individual treatments, compared to the CTL. Also, leaves of drought-stressed plants (individually and combined) presented higher hydrogen peroxide content than those of the CTL, while in roots, this ROS was enhanced with Cu exposure. Stress combination led to a greater oxidative damage, evaluated through lipid peroxidation, with no differences being detected in individual treatments, compared to the CTL. In terms of antioxidant response, proline levels were boosted by drought (individually and combined) and glutathione content was greater in plants under combined stress, compared to the CTL. Overall, plants subjected to a combination of Cu and drought triggered a differential oxidative response in comparison with each individual stress.
POG PP 2.13:
The herbicidal potential of eucalyptus leaves is related to oxidative bursts and subcellular damage – a case study in a model weed (Portulaca oleracea L.)
Mafalda Pinto | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
Climate change is aggravating the frequency and intensity of diseases and pests, including weeds’ proliferation. To counteract this, farmers are dependent on synthetic herbicide application. However, the generalized use of these agrochemicals has been accompanied by significant environmental impacts, inducing non-target toxicity in different biota, including crops. As the development of new eco-friendly solutions to reduce herbicides’ footprint while simultaneously controlling weed growth is urgently needed, the targeted use of the bioactivity of eucalyptus (Eucalyptus globulus Labill.) leaves to inhibit the growth of neighbor plants can constitute an effective approach. To test this hypothesis, different percentages (0, 1, 5, and 10% m/m) of dried and fresh leaves of young eucalyptus trees were incorporated in OECD soil, where purslane (Portulaca oleracea L.) seeds were sown. Over 5 weeks, the germination rate and weed growth were monitored. Results revealed that the incorporation of dried leaves at 10% had the highest herbicidal potential, hampering seed germination by 63% and seedling growth by 70%. To unravel the mode-of-action of this biocide in the early germination steps, purslane seeds were placed in Murashige and Skoog medium containing 0 and 250 g/L of an aqueous extract prepared with dried eucalyptus leaves. After 5 days, radicles and cotyledons were processed for ultrastructural analysis and reactive oxygen species (ROS) in vivo detection (superoxide anion and hydrogen peroxide) by confocal microscopy. The presence of the eucalyptus-based biocide impaired seed germination, corroborating soil-based assays, and led to marked subcellular disorganization and deposition of lipid vesicles in both organs. These changes were strictly related to an oxidative burst, with increased ROS accumulation in both purslane radicles and cotyledons. Overall, these results point to the efficiency of eucalyptus leaves to be used as a natural herbicide, capable of inhibiting weed germination and growth, inducing ROS overproduction and substantial changes in cell ultrastructure.
POG PP 2.14:
Revealing the nature of RCD1 nuclear bodies
Richard Gossens | Helsinki University | Finland
Plants' nuclei harbour various small membraneless compartments called nuclear bodies (NBs). These allow spatial separation of the nuclear compounds and create local biochemical environments favouring specific processes.
RCD1 is a nuclear protein that can interact with over 35 transcription factors and receive various retrograde stress signals. This makes RCD1 a very capable hub protein for integrating stress signals and orchestrating a balanced response. RCD1 localises to visually distinct NBs whose formation is enhanced by stress. The protein comprises three domains separated by intrinsic disorder regions (IDRs). We recently showed that the N-terminal WWE domain can bind the post-translational modification poly(ADP-ribose)(PAR) and that this binding is responsible for the formation of nuclear bodies. Following the WWE domain is an inactive PAR polymerase (PARP)-like domain, its function has hitherto been unknown. The C-terminal RST domain is responsible for transcription factor binding.
We generated a full-length RCD1-3xVenus line and three deletion lines omitting one domain at a time. By studying these, we observed distinct NB populations, and their size and number are dependent on both stress and on which domains were conserved. To reveal the composition of these NBs we exploited these deletion lines using co-immunoprecipitation followed by mass spectrometry. This approach indicates that the WWE domain, and thus PAR-binding, is associated with pre-mRNA splicing machinery while both the PARP-like domain and RST domain are associated with proteins involved in translation and proteasomal degradation. Therefore, RCD1 may be able to tailor alternative splicing, protein translation and proteasomal degradation to re-establish homeostasis during stress.
POG PP 2.15:
Transcriptomics analysis of the roles of a specific glucose-6-phosphate dehydrogenase in oxidative stress responses
Lug Trémulot | Université Paris-Saclay, Institute of Plant Sciences Paris Saclay (IPS2), Gif-sur-Yvette, France | France
Catalase-deficient plants such as the Arabidopsis cat2 mutant are useful systems to study responses to intracellular oxidative stress. In certain conditions, cat2 shows lesions on the leaves and accumulation of typical pathogenesis-related factors such as salicylic acid (SA) and SA-dependent gene expression. All these downstream effects of oxidative stress can be prevented by the sid2 mutation, which causes deficiency in the isochorismate synthase 1 (ICS1)-dependent pathway of SA synthesis.
NADPH is a key redox carrier in cells and can play multiple roles in reactive oxygen species (ROS) homeostasis and signaling. NADPH is required both for antioxidant metabolism (e.g., supporting the ascorbate-glutathione pathway) and for pro-oxidant ROS signaling (e.g., NADPH oxidase functions). Several types of enzyme can produce NADPH from the oxidized form, NADP+, and each of these enzymes is encoded by several genes. To explore specific roles of NADPH-producing enzymes in oxidative stress signaling, multiple loss-of-function mutants have been crossed with cat2. One of the double mutants obtained, cat2 g6pd5, in which a cytosolic glucose-6-phosphate dehydrogenase activity is compromised, shows much decreased oxidative stress responses, notably an absence of lesions on the leaves and weakened SA biosynthesis and signaling.
To explore how the g6pd5 mutation produces these effects, several approaches are being used, including transcriptomic comparison of cat2 g6pd5 and cat2 sid2. Mutants for NADPH-dependent pathways (ascorbate-glutathione pathway and NADPH oxidases) have also been crossed with cat2 to establish whether their transcriptomic signatures are similar to that observed in cat2 g6pd5. This will help to decipher the potential roles of a specific NADPH-producing respiratory enzyme in determining the outcome of oxidative stress of intracellular origin.
POG PP 2.16:
Small paraquat resistance proteins modulate paraquat and ABA responses and confer drought tolerance to Arabidopsis.
Prof. László Szabados | Biological Research Center | Hungary
Several small peptides have recently been described to modulate responses to stress conditions. The Small Paraquat resistance protein (SPQ) of Lepidium crassifolium has previously been identified due to its capacity to confer paraquat resistance to overexpressing transgenic Arabidopsis plants. Here we show, that overexpression of the closely related but previously unknown Arabidopsis SPQ can also enhance resistance to paraquat, while the knockout Arabidopsis mutant is slightly hypersensitive to this herbicide. Overexpression of SPQs enhanced sensitivity to abscisic acid (ABA), while the knockout spq1 mutant was less sensitive to ABA. Both Lepidium and Arabidopsis-derived SPQs could improve drought tolerance by reducing water loss, stabilizing photosynthetic electron transport, reducing oxidative damage and enhancing plant viability in a water-limited environment. Enhanced drought tolerance of SPQ overexpressing plants could be confirmed by characterizing parameters of growth, morphology and photosynthesis using an automatic plant phenotyping platform with RGB and chlorophyll fluorescence imaging. Our results suggest that SPQs can be regulatory small proteins connecting ROS and ABA regulation and through that influence responses to certain stresses.
POG PP 2.17:
Aldo keto reductases: New players in nitric oxide homeostasis
Dr. Patrick Treffon | University of Massachusetts Amherst | United States
Nitric oxide (NO) is a short-lived gas that acts as a signaling molecule in all higher organisms, including
plants. Despite the involvement of NO in multiple plant processes, including germination, root growth and
fertility, a basic understanding of the mechanisms by which NO exerts its effects is lacking. NO and its
derivatives impact these physiological processes through reversible S-nitrosation of critical protein
cysteines. In cells, regulation of NO-levels is predominantly achieved by reaction of reactive nitrogen
species (RNS) with glutathione (GSH), thereby forming S-nitrosoglutathione (GSNO), a principal NO
reservoir. Mutation of Arabidopsis thaliana S-nitrosoglutathione reductase (GSNOR; hot5-2) leads to
higher intracellular concentrations of S-nitrosothiols, confirming that the GSNOR reduction of GSNO is a
major route of GSNO catabolism in plants and other eukaryotes. We demonstrate in Arabidopsis that
absence of GSNOR results in differential regulation of proteins involved in chlorophyll metabolism, the
general stress response and photosynthesis. In addition, our proteomic analysis identified a significant
increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9.
Since specific AKRs have been linked to NO metabolism in mammals, we expressed and purified
Arabidopsis AKR4C8 and 9 and close homologues AKR4C10 and 11 and determined that they have
NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Plants lacking
GSNOR also show increased activity of NADPH-dependent GSNO reduction, consistent with increased
AKR activity. Taken together, these data define a new, NADPH-dependent component of NO
metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in
plants and other organisms.
POG PP 2.18:
Evolution and diversification of the NADPH oxidase family in plants
Julian Ingelfinger | Technical University Kaiserslautern | Germany
Reactive oxygen species (ROS) and redox signalling are evolutionary old and likely predated the origin of land plants. To specifically produce superoxide, plants possess enzymes of the NADPH-oxidase family, the respiratory burst oxidase homologs (RBOH). RBOHs are transmembrane enzymes that transfer electrons from NADPH across the plasma membrane to oxygen, producing superoxide in the apoplast. Apoplastic ROS regulate polar growth as well as stress responses. During land plant evolution the RBOH family evolved several isoforms with neo- and sub-functionalisation. However, it is yet unknown which photosynthetic organism were the first to possess a RBOH and which function that ancestral RBOH had. Available transcriptomic and genomic data suggest that RBOHs were already present in streptophyte algae. Using streptophyte algae, the model moss Physcomitrium patens as well as the model flowering plant Arabidopsis thaliana, we aim to unravel the ancestral function of RBOH isoforms. To monitor the balance between ROS generation and detoxification in vivo, we use redox sensitive GFP-based biosensors (roGFP2).
POG PP 2.19:
Mj-MSP18 effector suppresses ROS production and plant immunity: Could BSK7 be the explanation?
Boris Stojilkovic | Department of Biotechnology, Ghent University, Coupure Links 653, Ghent 9000, Belgium | Belgium
Plant-parasitic nematodes produce effectors to overcome plant immunity and finetune plant cellular processes. Many effectors have been shown to influence plant immunity to support nematode survival, but their direct targets and exact mode of action are still poorly understood. Identifying protein-protein interactions and newly formed complexes with effectors is crucial for understanding this cross-kingdom network. On the other side, it has been shown that host-derived ROS (reactive oxygen species) production plays a huge role in plant basal defense against nematodes.
Using the proximity labeling technique (TurboID-MS), we have identified tomato proteins involved in various cellular processes interacting with M. javanica effectors. Among those, the Mj-MSP18 effector, whose homolog has been shown to suppress cell death, was found to interact with tomato Sl-BSK7 potentially, a homolog of arabidopsis BSK7 (BRASSINOSTEROID-SIGNALLING KINASE7), serine/threonine kinase that acts as a positive regulator of brassinosteroid (BR) signaling downstream of the receptor kinase BRI1. To examine the influence of Mj-MSP18 on downstream signaling, we performed RNA-seq upon overexpression of Mj-MSP18 in tomato hairy roots and found downregulation of plant defense and response to hydrogen peroxide-related genes. Moreover, many downregulated genes have been previously reported to play a role in brassinosteroid-mediated resistance against root-knot nematodes. Furthermore, we confirmed a significant decrease in ROS production on a biochemical level upon transient expression of Mj-MSP18 in flagellin22 treated N. benthamiana leaves. Additionally, we depicted its role in suppressing plant cell death and callose deposition, one of plant immunity's main markers.
We hypothesized that differentially expressed genes are a likely consequence of MSP18 interaction with Sl-BSK7 and suppression of downstream signaling of BR receptor and its signaling transduction with FLS or other PAMP receptors leading to suppression of ROS, basal defense, and attenuation of host resistance to the nematode infection.
POG PP 2.20:
Bioactivity of rush-specific (Juncus sp.) compounds on antioxidant defence mechanisms of Arabidopsis thaliana
Dr. Ágnes Szepesi | University of Szeged | Hungary
The monocotyledonous rush species (Juncus) are mainly perennial plants, but some representatives are annual. Several Juncus plants are used in the Traditional Chinese Medicine for the treatment of numerous diseases. Juncaceae species produce different types of secondary metabolites e.g. phenantrenes, flavonoids, triterpenes and steroids, which can be very promising for the modern medicine as well. These natural compounds possess antiproliferative, anticancer, antimicrobial, anti-inflammatory, antioxidant, cellular protective and antialgal effects. However, these compounds have not been tested on antioxidant defense system of higher plants. Here, we showed the first bioactivity tests of phenantrene-like compounds, effusol and juncusol isolated from J. gerardii on Arabidopsis thaliana seedlings. Tested concentrations of phenantrene deterred the growth of A. thaliana. However, plants treated with effusol and juncusol did not show negative effects at these concentrations, influencing the antioxidant defense system of plants. Our results could help us to decipher the role of phenanthrene-like compounds in plant growth and development. This work was supported by OTKA FK129061 grant and NKFIH OTKA K 128963.
POG PP 2.21:
Data mining of potential carbonylation in the olive tree pollen and its physiological implications.
Dr. Juan de Dios Alché | Estación Experimental del Zaidín (CSIC) | Spain
The olive tree is an important crop in the Mediterranean area. The study of pollen biology is an essential task for plant improvement and to guarantee efficient fertilization and proper yield. Among the mechanisms modulating gene expression, different posttranslational modifications have been described. Carbonylation represents one of these modifications clearly indicative of the presence of oxidative phenomena derived from the presence of ROS and RNS. The present work shows represents an in silico prediction of the olive pollen carbonylome, obtained after using the iCarPS software tool with an experimentally determined proteome already described in the literature as the target. Bioinformatic prediction shows the presence of a large number of proteins prone to carbonylation, which has been experimentally confirmed in the present work after using an OxyblotTM kit in samples electrophoretically separated. Implications of this PTM on pollen biology have been discussed based in the results obtained.
This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).
POG PP 2.22:
Ferritin improves aluminium tolerance in chickpea by regulating the formation of reactive oxygen species
Prof. Dr. Bhumi Nath Tripathi | Indira Gandhi National Tribal University | India
The severity of aluminum (Al) toxicity on agricultural productivity in acidic soil has spurred vast research studying Al tolerance in plants. The efflux of organic acids e. g. citrate, malate, and activation of their transporters have been proved as the key mechanism of Al tolerance. However, like other abiotic stresses, Al toxicity has great potential to generate oxidative stress in the plant cell. But research elucidating the effective management of oxidative stress in the plant during Al toxicity is not available. The present study demonstrates the development of Al tolerance in chickpea by controlling oxidative stress. Two contrasting genotypes of chickpea e. g. RSG 974 (Al tolerant) and RSG 945 (Al sensitive) were used as test plants. Al-tolerant genotype (RSG 974) showed lesser inhibition of root growth as well as lower oxidative damages, measured in terms of the accumulation of H2O2 and lipid peroxidation compared to the Al-sensitive genotype (RSG 945). Subsequently, DDRT-PCR results showed the differential expression of two genes, chitinase and ferritin in chickpea genotypes after the treatment of 1.0 mM Al. Further, Q-PCR analyses confirmed the Al-responsive expression of chitinase in the Al-tolerant chickpea genotype, whereas, the expression of ferritin was found to be constitutively higher in the Al-tolerant genotype compared to Al-sensitive chickpea. The biochemical data showed that the higher expression of ferritin in Al-tolerant chickpea genotypes in the present study is associated with the efficient sequestration of free iron responsible for generating reactive oxygen species. Whereas, aluminium sensitive genotype of chickpea (RSG-945) due to negligible expression of ferritin could not efficiently control the free form of Fe and hence failed to avoid the consequences and thereby became more susceptible to Al toxicity. Further experiments are underway to provide the mechanistic details of managing oxidative stress in chickpea by ferritin to alleviate Al toxicity.
POG PP 2.23:
Systematic monitoring of 2-Cys peroxiredoxin-derived redox signals unveiled its role in attenuating carbon assimilation rate
Dr. Nardy Lampl | The Hebrew University of Jerusalem | Israel
Identifying the intrinsic factors that regulate leaf photosynthetic rate may pave the way toward developing new strategies to enhance carbon assimilation. Transmission of reductive and oxidative cues from the photosynthetic electron transport chain to redox regulatory protein networks plays a crucial role in coordinating photosynthetic activities. The tight balance between these two signals dictates the cellular response to changing light conditions. While the role of reductive signals in activating chloroplast metabolism is well-established, the role of their counterbalanced oxidative signals is still lacking. By developing 2-Cys peroxiredoxin-based genetically encoded biosensors, into Arabidopsis thaliana chloroplasts, we monitored the dynamic changes in photosynthetically-derived oxidative signaling. We showed that chl-roGFP2-PrxΔCR oxidation states reflected the similar oxidation patterns of endogenous 2-Cys peroxiredoxin under varying light conditions. We also demonstrated the induction of 2-Cys peroxiredoxin-dependent oxidative signals, throughout the day, under varying light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals. Furthermore, we unraveled the simultaneous activation of reductive and oxidative signals during photosynthesis induction phase and showed that 2-Cys peroxiredoxin activity attenuates carbon assimilation rates, demonstrating the restrictions imposed on photosynthetic performance by oxidative signals.
POG PP 2.25:
Proline accumulation under glyphosate toxicity - a signal of tolerance or susceptibility?
Dr. Cristiano Soares | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
Glyphosate (GLY) is the most used herbicide worldwide, and its cumulative use has been resulting in the contamination of agricultural soils, potentially harming non-target organisms, like crops. Despite GLY's herbicidal activity being not directly related to oxidative bursts, research suggests that a disruption in the redox equilibrium is frequently an unintended result of GLY exposure. Thereby, plants must orchestrate a fine regulation of their antioxidant mechanisms to prevent oxidative stress. Once exposed to GLY, plants experience an overaccumulation of proline (Pro), although this does not seem to be accompanied by a tolerance response. Therefore, the primary question driving this research is whether Pro overaccumulation in response to GLY is a tolerance mechanism or a stress signal. Firstly, Pro content was determined in plants growing on a nutritive medium with increasing GLY concentrations (0.5, 1, and 2 mg/L) for 14 days, to prove that GLY leads to a Pro increase. Then, Arabidopsis thaliana T-DNA insertional mutant lines for genes involved in the Pro pathway (P5CS1 and ProDH) were used to unravel the role of this amino acid in GLY-induced stress. After their functional characterization, p5cs1-1, p5cs1-4, and prodh mutants were used, along with wild-type plants, in an in vitro experiment. After 14 days of exposure to GLY (1 mg/L), a decrease in seedlings’ biomass upon herbicide exposure was observed in all genotypes, this being followed by an overaccumulation of Pro. The data also showed that prodh seedlings were the most sensitive, reaching growth inhibition values of 80%, and those that accumulated the highest levels of proline in response to GLY. Overall, these findings suggest that Pro overaccumulation under GLY exposure is related to stress sensitivity rather than stress tolerance. To get a wider picture of the compensatory mechanisms involved, the oxidative metabolism of these genotypes under GLY is currently being studied.
POG PP 2.26:
PGRL1 redox states alleviate photoinhibition in Arabidopsis during step changes in light intensity
Dr. Amit Kumar Chaturvedi | Weizmann Institute of Science | Israel
Non-motile plants have evolved regulatory mechanisms to maintain homeostasis for optimal growth. Responses to environmental changes in light are particularly important not only during the diurnal transition from night to day but also to react to light changes caused by passing clouds or by the wind. Thioredoxins rapidly orchestrate redox control during environmental change by modifying cysteine residues. Here, we assign a function to regulatory cysteines of PGRL1A, a constituent of the ferredoxin-dependent cyclic electron flow (Fd-CEF) pathway and show their role in the regulation of proton motive force (PMF) and nonphotochemical quenching (NPQ). During step increase of low light intensity (10-60 µE*m-2*s-1), the intermolecular disulfide of the PGRL1A 59-kDa complex is reduced transiently within seconds to the 28 kDa form. In contrast, step increases to higher light intensity (60-600 µE*m-2*s-1) stimulated a stable partially reduced redox state in PGRL1A. Measurements of NPQ, PMF and resultant photosynthetic controls Y(ND) and Y(NA) were found to correlate with the redox state of PGRL1A during step increases in light intensity but not in PGRL1mutant plants pgrl1ab or PGRL1A cysteine mutant (PGRL1AC1,2A). Continuous light regimes did not affect mutant growth; however, fluctuating regimes of light intensity showed significant growth reduction in the mutants. Inhibitors of photosynthesis placed control of the PGRL1A redox state as dependent on the penultimate ferredoxin redox state that fuels reducing equivalents to the large set of chloroplasts thioredoxins. Our results showed that redox state changes in PGRL1A are crucial to the optimization of photosynthesis and are regulated by the photosynthetic electron flux.