POG PP 1.1:
Intercropping and sequential cropping between tomato and halophytes: physiological and biochemical responses under saline conditions
Gregorio Barba Espín | CEBAS-CSIC | Spain
Halophytes are known to accumulate excess salts in tissues, removing them from the immediate environment. This makes them suitable candidates for haloremetiation. In this two-phase experiment, we explored the feasibility of intercropping tomato (Solanum lycopersicum var. Sargento) with the halophyte Arhtrocaulon macrostachyum, native of South East Spain, trying to mitigate the negative effects of saline soil while providing a value-added crop.
Two consecutive greenhouse experiments, from March to July 2021 and from October 2021 to February 2022, were conducted under soil Na+ and Cl- contents of 950 ppm and 1500 ppm, respectively. Plots were arranged in randomised block designs with three replicates. In the first season, three types of plots – halophyte and tomato in monocultures and mix cultivation – were arranged, whereas in the second season tomato was additionally cultivated where halophyte was previously grown (sequential cropping).
Overall, intercropping and sequential cropping affected the activity of antioxidants enzymes in leaf extracts. This was reflected on an enhanced accumulation of reactive oxygen species, which may lead to the establishment of a moderate oxidative stress. In parallel, changes on chlorophyll fluorescence parameters were recorded. In this sense, non-photochemical quenching (NPQ) and electron transport rate (ETR) were higher in intercropping and sequential cropping than in monoculture. With respect to the halophyte, both leaves and roots showed higher Na and Cl than in monoculture, while in tomato the opposite behaviour occurred. Intercropping did not affect tomato production. However, sequential cropping significantly increased (up to 20%) both tomato number and weight. On the other hand, sequential cropping decreased soluble solids (measured as °Brix) and acidity (as total acidity to citric acid equivalents) for tomato in comparison to both tomato in monoculture and in intercropping. Further experiment replications will be conducted, which will also involve valorization of the halophyte for ulterior uses.
POG PP 1.2:
Cysteine-based redox switches in plant stress response
Dr. Jingjing Huang | Ghent University | Belgium
The hasher environments resulted by climate change cause oxidative stress to plants, triggering reactive oxygen species (ROS) production in plant cells, which are potent signaling molecules prone to activate defense responses. Relatively stable ROS, such as hydrogen peroxide (H2O2), can provoke reversible and irreversible oxidative post-translational modifications (Ox-PTMs) on protein cysteine (Cys), which might act in diverse signaling pathways in plant stress adaption. Protein Cys thiols (–SH) are very susceptible to H2O2. The initial reaction of H2O2 with –SH forms sulfenic acid (–SOH) that is intrinsically unstable and an intermediary en route to other OxiPTMs, such as the relatively more stable sulfinic (–SO2H) and sulfonic (–SO3H) acids, which are consider as overoxidation. The –SO3H formation is irreversible, whereas –SO2H can be recycled in an ATP-dependent manner by sulfiredoxin (Huang et al. 2018; Willems et al. 2021).
To get insight into Cys-mediated redox switches in plants, we have generated an unprecedented view on the –SOH landscape using variou proteomic approaches that facilitate further functional redox studies (Huang et al. 2019; Wei et al. 2020). To better understand the protection mechanism from Cys overoxidations, our current ongoing work also focuses on –SO2H and its reduction enzyme, sulfiredoxin. We aim to uncover the Cys-based redox switches in plants under oxidative stresses. The identified Cys-based redox mechanisms could be extrapolated and contribute to optimize crops and vegetables to be more resilient against climate change. Here, the general strategy for studying Cys oxidation will be outlined, and several examples for functional studies including ongoing work will be presented.
POG PP 1.3:
Integrative inference of transcriptional networks in Arabidopsis yields novel ROS signalling regulators
Xiaopeng Luo | Belgium
Gene regulation is a fundamental process during plant growth and development, but is also important for plant responses to external stimuli. Gene regulatory network plays important roles in different physiological responses and pathways to help plant adapt to the environments. However, our global knowledge about the complexity of TF control for different genes and biological processes is incomplete. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF binding and expression-based regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions) in this work. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines (MV, H2O2 or 3-AT). In conclusion, the presented iGRN offers a high-quality starting point to give us a better understanding for gene regulation in plants by integrating different experimental data types at the network level.
POG PP 1.4:
THE SAGA COMPLEX SUBUNIT GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) IS REDOX SENSITIVE
Dr. Pavel Kerchev | Mendel University Brno | Czech Republic
The evolutionary conserved Spt–Ada–Gcn5–Acetyltransferase (SAGA) complex is a transcriptional coactivator that regulates a myriad of cellular processes through modulation of chromatin structure and transcription. As a part of its histone acetylation module, the histone acetyltransferase GENERAL CONTROL NONDEREPRESSIBLE 5 (GCN5) acetylates lysine 14 of histone 3 (H3K14) in the promoter regions of its target genes. GCN5 has been implicated in stress responses and development processes thus functioning at the crosstalk between environmental and stress programs. Intriguingly, despite its profound impact on gene expression, the molecular mechanisms regulating the acetylation activity of the SAGA complex are still unknown. Here, we report that GCN5 is sulfenylated at an evolutionary conserved cysteine residue and this modification is implicated in mounting an effective stress response. Complementation of the gcn5 mutant with a GCN5 variant carrying a cysteine to serine mutation reverted the dwarfed gcn5 phenotype under control conditions, whereas it did not impact the sensitivity to oxidative stress observed in gcn5 mutant plants. Our results suggest that GCN5 is an important redox sensitive component of the SAGA complex that might play a role in novel molecular mechanisms integrating redox signaling and chromatin remodeling.
POG PP 1.5:
Carotenoid biosynthesis contributes to the ascorbate-glutathione cycle regulation to protect against ROS accumulation in microalga Euglena gracilis
Dr. Shun Tamaki | RIKEN | Japan
The antioxidants, ascorbate and glutathione, which are regenerated by the ascorbate-glutathione cycle, and carotenoids, are required for oxidative stress defense in photosynthetic organisms. The euglenophyte Euglena gracilis is a model microalga, which is used for both biological studies and industrial applications. Previous study has shown that treatment of E. gracilis with carotenoid biosynthesis inhibitor, norflurazon, suppresses photo-induction of ascorbate peroxidase enzyme (APX), suggesting the functional relationship between carotenoids and the ascorbate-glutathione cycle in this alga. In this study, using RNAi-mediated knockdown cells of carotenoid biosynthetic gene lycopene cyclase (EgLCY), we investigated the effects of suppressed carotenoid biosynthesis on the ascorbate-glutathione cycle and oxidative stress status in E. gracilis.
The KD-lcy cells, which were introduced double-stranded RNA, showed colorless appearance and 81% decrease in the total carotenoid contents compared to wild-type cells. The APX and superoxide dismutase (SOD) activities of KD-lcy cells decreased by 52% and increased 7.9 times, respectively, compared to wild-type cells. Among the ascorbate-glutathione cycle, the dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MADR) activities of KD-lcy cells increased by 77 and 45%, respectively, compared to wild-type cells and their glutathione reductase (GR) activity decreased by 29%. Correlating with the altered enzyme activities of the ascorbate-glutathione cycle enzymes, the ascorbate contents of KD-lcy cells increased by 55%, whereas their glutathione contents decreased by 38% compared to wild-type cells. The imaging analysis using H2O2-specific fluorescent reagent demonstrated that KD-lcy cells accumulated significantly higher level of H2O2 than that of wild-type cells. These results reveal that carotenoid biosynthesis contributes to chloroplast development and the ascorbate-glutathione cycle homeostasis, resulting oxidative stress defense in E. gracilis. To our knowledge, this study clearly reports for the first time the possible relationship between major antioxidant systems carotenoid biosynthesis and the ascorbate-glutathione cycle in photosynthetic organism.
POG PP 1.6:
Different oxidative pattern in sensitive and resistant Amaranthus palmeri populations treated with herbicides inhibiting amino acid biosynthesis
Mikel Vicente Eceiza | Institute for Multidisciplinary Research in Applied Biology (IMAB), Public University of Navarre | Spain
Glyphosate, the most used herbicide worldwide, inhibits the 5-enolpiruvyl-3-phosphate synthase (EPSPS) in the shikimate pathway. Acetolactate synthase (ALS) inhibitors are a diverse herbicide group that inhibit ALS in the branched-chain amino acid biosynthesis pathway. Besides their different enzymatic target, glyphosate and ALS inhibitors trigger common physiological effects, including oxidative stress. However, the linkage between EPSPS/ALS inhibition and oxidative stress is not completely elucidated. Additionally, the massive usage of these herbicides has led to the development of weed resistant populations, as in Amaranthus palmeri.
Physiological effects of herbicides in resistant populations are poorly studied. The objective is to get new insights in the oxidative stress triggered by glyphosate and ALS inhibitors comparing sensitive and resistant plants with resistance mechanisms related to the target enzyme. To this purpose, glyphosate-resistant and ALS inhibitor-resistant populations and their sensitive reference populations were grown and treated with different doses of glyphosate or the ALS inhibitor nicosulfuron, respectively: untreated, field rate and 3 times field rate. Hydrogen peroxide content and gene expression levels of the antioxidant enzymes superoxide dismutase (CuZnSOD) and glutathione reductase (GR1, GR2) were measured as oxidative stress parameters.
All herbicide treatments were lethal for sensitive individuals, while resistant individuals survived. Early upon herbicide treatment, H2O2 highly accumulated but only in sensitive plants. Superoxide dismutase gene expression was induced in both sensitive and resistant populations after exposure to both herbicides. The GR1 expression level increased with glyphosate, especially in the sensitive population; while it did not change with nicosulfuron. The GR2 expression was unaltered by herbicide treatments.
In conclusion, the increase in antioxidant gene expression induced by glyphosate and ALS inhibitors in sensitive plants was not enough to avoid H2O2 accumulation in contrast to resistant populations. Probably their resistant physiology and mild increase in CuZnSOD and GR1 gene expression is sufficient to avoid oxidative stress.
SFRR-E PP 1.7:
Non-enzymatic S-nitrosation and denitrosation of proteins by variation of superoxide/nitric oxide ratio – implications for prevention of sulfoxidation-dependent enzyme inactivation during ischemia/reperfusion
Prof. Dr. Andreas Daiber | University Medical Center Mainz | Germany
Reactive oxygen and nitrogen species play a key role for the development of cardiovascular, metabolic and neurodegenerative disease, but they are also involved in cellular functions via redox signalling. We have previously identified an efficient mechanism of S-nitrosation by low levels of nitric oxide and superoxide (3:1 ratio) with potential formation of N2O3. Here, we elucidated whether S-nitrosation (as observed under hypoxic conditions) could prevent sulfoxidation and thereby oxidative inactivation of enzymes by superoxide/hydrogen peroxide (as observed during reoxygenation). We found that increasing concentrations of xanthine oxidase/hypoxanthine caused conversion of S-nitrosoglutathione (GSNO) to reduced glutathione (GSH) up to a certain concentration of xanthine oxidase, indicating that superoxide can induce denitrosation of GSNO. This finding was unexpected since in the presence of excess superoxide one would not expect regeneration of reduced GSH from GSNO. Also, the observed substantial dihydrorhodamine oxidation that was prevented by uric acid and tyrosine nitration of albumin during denitrosation of GSNO by superoxide, clearly pointed to intermediary formation of peroxynitrite. As a proof-of-concept, we demonstrate that isocitrate dehydrogenase (ICDH2) can be S-nitrosated / inactivated by spermine NONOate and denitrosated / partially reactivated by superoxide formed by xanthine oxidase. In summary, we propose that S-nitrosation of (mitochondrial) proteins during ischemia represents a protective mechanism to prevent irreversible overoxidation of thiols during the reperfusion phase and to re-establish reduced thiol state in (mitochondrial) key enzymes of energy metabolism and cell survival. Wide-spread mitochondrial protein S-nitrosation may represent a central feature of the protective preconditioning effects of nitric oxide.
POG PP 1.8:
The redox environment differentially regulates autophagy in leaves and roots of Arabidopsis thaliana during cadmium stress
Isabeau Vanbuel | Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium | Belgium
Pollution of soils with metals such as cadmium (Cd) inhibits plant growth, thereby hindering their economic validation. Cadmium induces an imbalance between reactive oxygen species (ROS) and antioxidants at the cellular level, which can damage macromolecules. Damaged cellular components can be engulfed by vesicles (i.e. autophagosomes) that transport them into the vacuole for nutrient recycling in a process called autophagy. Autophagy is increasingly put forward as a protective mechanism during various stresses that may be intertwined with the redox environment. Therefore, this research aims to investigate autophagy in leaves and roots of Arabidopsis thaliana exposed to Cd and its connection to the Cd-induced oxidative challenge.
One important protein that is involved in autophagosome formation is ATG8, which is encoded by nine isoforms in A. thaliana. At the transcript (RT-qPCR) and protein levels (western blot), A. thaliana plants exposed to 5 μM Cd for 24 h in hydroponics show an increased expression of ATG8. Confocal microscopy analyses are currently performed in Cd-stressed GFP-ATG8 reporter lines to further confirm autophagy induction. To investigate the link between autophagy and the redox environment during Cd stress, the expression of all ATG8 isoforms was compared between wild-type and glutathione (GSH)-deficient cad2 plants. Overall, no clear difference could be observed between both genotypes under control conditions. However, an overall lower induction could be observed for Cd-responsive ATG8 isoforms in the leaves of mutant plants following Cd exposure. In contrast, the overall expression level in the roots was rather higher in Cd-exposed mutants compared to wild-type plants, which was mainly attributed to ATG8E. This indicates that the redox environment differentially affects ATG8 expression and potentially autophagy in both plant organs during Cd stress.
POG PP 1.9:
Characterization of the Arabidopsis RBPome in oxidative stress conditions
Dr. Zhicheng Zhang | VIB-UGent Center for Plant Systems Biology, Ghent University | Belgium
Cellular redox signaling is triggered by accumulation of various reactive oxygen species (ROS) that integrate with other signaling cascades to enable plants to ultimately respond to (a)biotic stresses. The identification of key regulators underlying redox signaling networks is therefore of high priority. We improved an mRNA interactome capture method that allows to systematically detect oxidative stress responsive regulators in the post-transcriptional gene regulation (PTGR) pathway. The protocol includes Arabidopsis cell cultures, setup of oxidative stress conditions, short-term exposure to UV irradiation, cell lysis, pull-down and purification of crosslinked messenger ribonucleoproteins, their mass spectrometric analyses and identification of the proteome by statistical analyses. As a result, a comprehensive inventory of the functional oxidative stress responsive RBPome (OxRBPome) is generated. Novel candidate proteins with previously unlinked functions to RNA biology, were further explored to pave the way towards new insights into PTGR processes in redox signaling. The obtained comprehensive view of the RBPome under oxidative stress provides us a more detailed insight and understanding of post-transcriptional regulatory processes in redox signaling in plants.
POG PP 1.10:
Analysis of the salt-stress dependent modulation of the redox network in rice
Dr. Michela Molinari | Università Campus Bio-Medico di Roma | Italy
Global climate change cause various adverse environmental conditions, such as drought, salinity, high temperature, and toxic metal accumulation, which affect plants growth and may affect food security. Rice (Oryza sativa) feeds more than one half of the world’s population and is the model system for monocotyledonous. However, rice is the most salt sensitive cereal crop. To ensure rice production and preserve the biodiversity, in salt-affected soils, is crucial to understand the stress-induced metabolic alterations in tolerant and resistant plants, in order to identify interesting traits for improving plant resilience toward unfavorable environmental conditions. We have investigated on the mechanisms underpinning tolerance towards salinity, focusing on root system of two rice varieties showing contrasting resistance to salt, Baldo and Vialone Nano. Plants have evolved several interconnected molecular pathways to defend themselves against different abiotic stresses. A common theme during plants’ stress responses and adaptation is the production of reactive oxygen species (ROS) and the redox signaling plays a pivotal role in determining plant tolerance and survival to stress. A detailed analysis of the salt stress-dependent modulation of the redox network is here presented. The different phenotypes observed after salt exposure in the two rice varieties is coherent with a differential regulation of cell cycle progression and cell death patterns observed at root level. Baldo showed a highly responsive antioxidative capacity, and different pattern of H2O2 accumulation compared to Vialone Nano. Moreover, glutathione metabolism was analyzed at transcriptional, post-transcriptional and post-translational level in both varieties. These results contribute to highlight the role of ROS and antioxidative pathways as a part of a complex redox network activated in rice toward salt stress.
POG PP 1.11:
Heat shock may impair non-host resistance of barley to Tobacco mosaic virus by inhibition of an early ROS (superoxide) burst
Dr. Lóránt Király | Plant Protection Institute, Centre for Agricultural Research, ELKH | Hungary
Tobacco mosaic virus (TMV) replicates in non-host barley at prolonged high temperatures (30 °C), especially if heat-exposed barley is infected by an adapted virus, e.g. Barley stripe mosaic virus (BSMV) (Dodds and Hamilton, 1972, Virology 59, 418). We have shown recently that non-host resistance to TMV in BSMV-infected barley can be also suppressed by heat shock pre-treatments (Király et al., 2021, FESPB-EPSO Plant Biology Europe Congress). In the present study we aimed at elucidating 1/ How heat shock influences non-host resistance of barley to TMV in absence of an adapted virus like BSMV, 2/ Does an early burst of reactive oxygen species (ROS) like superoxide (O2.-) play a role in the non-host resistance of barley to TMV?
In TMV-inoculated barley (cv. Ingrid), heat shock pre-treatments (30 °C for 3 hours, before TMV inoculation; 49 °C for 20 seconds, at 2 hours before TMV inoculation) resulted in significantly (50-100%) higher TMV accumulation, compared to non-pretreated controls kept at constant 20 °C (assayed by RT-qPCR). It seems that heat shock may impair non-host resistance of barley to TMV even in absence of an adapted virus like BSMV. Expression of a NADPH oxidase gene (HvRBOHF2) governing superoxide production and host disease resistance, and HvSOD1 (superoxide dismutase) and HvBI-1 (BAX-inhibitor), genes encoding for an antioxidant and a cell death regulator, respectively, displayed an inverse correlation with TMV levels, even at relatively late time points (1 to 7 DAI). This implies a pivotal role of these defense-related genes in maintaining non-host resistance to TMV. Furthermore, we found that an early (2 HAI) burst of superoxide is essentially absent in TMV-infected barley exposed to heat shock pre-treatments (assayed by nitro blue tetrazolium chloride /NBT/ staining), pointing to the role of ROS (superoxide) in non-host resistance of barley to TMV.
Acknowledgement: A grant from NKFIH (K128868)
POG PP 1.12:
Exposure to hydrogen peroxide from beverages and food
Prof. Dr. Izabela Sadowska-Bartosz | University of Rzeszow | Poland
It has been reported that hydrogen peroxide is generated in such beverages as tea and coffee, due to autoxidation of polyphenols present in the extracts. As polyphenols are present also in other beverages and food of plant origin, this study was aimed at checking whether H2O2 can be present in other commonly consumed products. We estimated the formation of hydrogen peroxide in wine, herbal infusions and cooked vegetables using the Xylenol Orange method, enhancing its specificity by assaying two parallel samples, one of them preincubated with catalase.
Variable low concentrations of hydrogen peroxide were found to accumulate in freshly opened red wines during 3-h incubation, from 0 to 6.6 µM, depending on the type and batch. Higher concentrations accumulated during 3-day storage of opened wines (up to ca 30 µM and ca 20 µM in a red wine and a white wine studied, respectively). Like in the tea, hydrogen peroxide was generated in infusions of medicinal herbs. From among 16 herbs studied, the highest concentrations of H2O2 were found in fresh 1% (w/v) infusions of the birch Betula pendula leaves (34.3±3.9 µM), of inflorescence of the lime Tilia cordata (25.9±1.2 µM) and of leaves of the plantain Plantago lanceolata (22.3±0.6 µM). Hydrogen peroxide was also found in the homogenates of cooked vegetables. The highest concentration of hydrogen peroxide in 1:2 (w/v) homogenates was found for the broad bean (73.4±9.0 µM) followed by broccoli (18.6±0.3 µM), onion (10.4±1.6 µM) and leek (10.0±0.3 µM) at pH of about 7; H2O2 concentrations were lower at lowered pH.
These results show that our nutritional exposure to hydrogen peroxide may be higher than estimated to date. These small concentrations of H2O2 do not seem to have deleterious health effects and may be even beneficial due to their bactericidal and virucidal action.
POG PP 1.13:
Thiol-based redox regulation of Arabidopsis Class II PABPs
Zeya Chen | Belgium
In eukaryotes, poly(A) binding proteins (PABPs) bind to the poly(A) tail of transcripts and thereby influence the RNA stability, nucleo-cytoplasmic transport and translation. Three members of PABPs (PABP2, 4 and 8) in Arabidopsis, which form the class II PABPs, play various roles in plant growth and development as loss-of-function double mutants (pabp2/4, pabp2/8 and pabp4/8) exhibit phenotypic abnormalities in leaf shape, flowering time and plant height . In previous redox proteomics studies, we charted the sulfenomic landscapes in Arabidopsis. There, we demonstrated that upon H2O2 stimulation of cells, class II PABPs underwent thiol-dependent oxidative post-translational modifications on Cysteine (Cys) residues, which reside in their RNA recognition motif (RRM) domains [2, 3]. Therefore, we hypothesized that Cys oxidation might affect the RNA binding activity of class II PABP. First, by means of Electrophoretic Mobility Shift Assays (EMSA) with recombinantly expressed proteins and biotinylated poly(A), we found that the poly(A) binding capacity of class II PABP proteins decreased under elevated H2O2 concentrations. Mutated isoforms in which the Cys residues were replaced with Serine residues, were insensitive to H2O2 dose-dependent decrease of poly(A) binding activity. In order to functionally characterize the thiol-based redox regulation of Class II PABPs, we are currently generating transgenic plants in which both wildtype and Cys mutant PABPs are ectopically expressed into double mutants (pabp2/4 and pabp2/8) for future phenotypic analysis under normal growth and oxidative stress conditions.
1. Gallie, D.R., Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development. Translation, 2017. 5.
2. Huang, J., et al., Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites. Proc Natl Acad Sci U S A, 2019. 116.
3. Wei, B., et al., Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter. Frontiers in plant science, 2020.11
POG PP 1.14:
Thallium induced increases on O2.-, H2O2, NO and H2S production, and morpho-physiological alterations in Ditrichia viscosa plants.
Dr. Francisco Espinosa | Universidad de Extremadura (NIF Q0618001B) | Spain
The alterations induced by the toxicity of thallium (Tl) in the roots and leaves of Dittrichia viscosa plants were determined. The plants were grown hydroponically with different concentrations of Tl (0, 10, 50 and 100 µM) during 7 days, a metal which reduces biomass production and growth, and the relative water content decrease. The leaves shown venal chlorosis. Tl is accumulated mostly in the roots, with the concentrations in the leaves being much lower. ICP-MS analysis showed that the K content decreased both roots and leaves. Fe, Cu, Mn concentrations was altered.
Chlorophyll a and b content declined, and the ratio chl a/chl b increased, but the carotenoid content increase (x2.2). The photosynthetic efficiency decrease for higher Tl concentrations (0.779, 0.731, 0.547 and 0.522 for control, 10 µM, 50 and 100 µM Tl, respectively). These results would indicate that Tl causes an alteration of the photosynthetic apparatus, probably by increasing ROS despite the increase in carotenoids.
Increases were observed in the amount of lipid peroxidation in roots, but not in leaves. The O2.-, H2O2, NO increase, specially in roots. The H2S content increases in leaves, but in roots only with the 10 µM Tl. The induced oxidative stress leads to a strong increase in the superoxide dismutase (SOD).
Tl induced alterations in ascorbate homeostasis, the AsA increase and DHA decrease. The ascorbate pool only decrease with 100 µM Tl. However, the glutation pool decrease depending of Tl concentrations. The GSH content decrease and almost all glutathione is GSH.
In summary, in Dittrichia the Tl accumulates more in roots than in leaves. Tl toxicity induces oxidative stress, with redox homeostasis imbalance. These alterations are greater in roots than in leaves, where the antioxidant system is more efficient.
Acknowledgments: This study was made possible thanks to the Junta de Extremadura/FEDER (GR21112).
POG PP 1.15:
Hydrogen sulfide (H2S) in horticultural plants: endogenous detection and its correlation with L-cysteine desulfhydrase (LCD) activity
María Ángeles Muñoz-Vargas | Spanish National Research Council (CSIC) | Spain
H2S has acquired great attention in plant research because it has signaling functions under physiological and stress conditions. However, the direct detection of endogenous H2S and its potential emission is still a challenge in higher plants. Using the fresh extract of different plant species with agronomical interest including pepper fruits, broccoli, ginger, and different members of the genus Allium including garlic, leek, welsh and purple onion, it was determined the endogenous H2S and its emission using an ion-selective microelectrode and a specific gas detector, respectively. The data show that endogenous H2S content range from pmol to μmol H2S · g-1 fresh weight whereas the H2S emission of fresh-cut vegetables was only detected in the different species of the genus Allium with a maximum of 9 ppm in garlic cloves. Additionally, it was characterized the activity and isozymes of the L-cysteine desulfhydrase (LCD), which is one of the main enzymatic sources of H2S, being the different species of the genus Allium which showed the highest activities. Using non-denaturing gel electrophoresis, the data indicated the presence of up to 9 isoenzymes different LCD isozymes from one in ginger to four in onion, leek, and broccoli. In summary, the data indicate a correlation between higher LCD activity with the endogenous H2S content and its emission in the analyzed horticultural species.
[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 1.16:
How can redox systems explain water stress tolerance in the grain legume Lathyrus sativus – preliminary results
Matilde SANCHES | VIB-UGent Center for Plant Systems Biology, Ghent University | Belgium
Given the current Climate Change scenario, a deeper understanding of abiotic stress resistance in crops is a priority towards securing food and feed supplies. Plants have evolved diverse strategies to avoid and/or cope with the increasingly frequent drought and flooding events and the various stresses associated with them, namely oxidative stress.
Studying reactive oxygen species (ROS) homeostasis within the context of water stress in plants is important, not only because antioxidant defense plays a central role in preventing the impacts of oxidative stress, but also, because complex and highly articulate signal transduction networks involving ROS are required for sensing and adapting to new environmental conjectures.
The legume Lathyrus sativus L. (grass pea) is of high economic importance for food and feed in Asian and African developing countries. Interest has been raising also in the Mediterranean region, where this crop is part of cultural heritage of more marginal areas, due to its outstanding robustness under adverse environmental conditions, like salt, temperature, and water stress, compared with other legume species.
The aim of this work is to unravel some of the molecular mechanisms underlying grass pea’ water and oxidative stress response by comparing tolerant and susceptible grass pea accessions, previously identified by an extensive phenotyping of a worldwide collection of germplasm of this species, evaluated under three water treatments (well-watered, waterlogging and water deficit).
Some preliminary metabolite quantification results are presented, namely of glutathione and ascorbate oxidized and reduced forms, as an attempt to unravel contrasting redox conjectures among susceptible and tolerant plants, subject to different water treatments.
Future biochemical and transcriptomic studies will deepen the understanding of the potential role of ROS in response mechanisms to water deficit and waterlogging in grass pea.
POG PP 1.17:
Polyamines-a novel molecular coordinator of nitro oxidative signaling in barley senescing leaf
Dr. Ewelina Paluch-Lubawa | Adam Mickiewicz University in Poznań, Poland, Department of Plant Physiology | Poland
Plant cells sense fluctuations in the levels of cellular polyamines (PAs), namely, putrescine (Put), spermidine (Spd) and spermine (Spm). PA cellular titer regulates biological processes, including senescence. Changes in PA concentration in properly functioning cells are unlikely. Induced senescence disturbs their homeostasis which leads to senescence-dependent metabolic changes. PA catabolism was proposed as process promoting mainly by H2O2 production. Here, we show that darkness-induced plant senescence is associated with changes in the PA levels together with the changes of signaling molecules such as nitric oxide (NO) and hydrogen peroxide (H2O2). Upon blocking Put catabolism, the senescing cells generated lesser amounts of NO and more of H2O2 and, in turn, accelerated senescence. We opine that a possible linkage exists between PAs and signaling molecules such as NO and H2O2. Thus, the balance in the triad network made of NO↔PA↔H2O2 signaling may be involved in re-programming metabolism and regulate the direction in which a plant may be ushered into - growth, senescence or cell death.
This work was supported by Polish National Science Centre in the frame of the project 2017/27/N/NZ9/02135 (EPL)
POG PP 1.18:
Dynamics of nitration phenomenon during dark-induced leaf senescence of Arabidopsis WT plants
Prof. Magdalena Arasimowicz-Jelonek | Department of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University | Poland
The study presents new insights into the role of nitric oxide metabolism in senescing Arabidopsis leaf defined as the dynamics of the nitration phenomenon. Detection of reactive oxygen and nitrogen species in dark-induced leaf senescence (DILS) model allows us to confirm that senescence-like phenomena are accompanied by a gradual decrease in NO emission and indicated a transient wave of peroxynitrite (ONOO–) formation on day 3 of DILS. To assess the metabolic fate of the time-dependent ONOO– bioavailability in individually darkened leaves (leaf 7 of the rosette), nitration levels at protein and nucleic acids level were determined on the following days of DILS (from day 1 to day 7). As evidenced by immunoassay the boosted ONOO– formation did not promote protein tryptophan nitration and progress of senescence depleted the pool of nitrotryptophan containing proteins. On the contrary, nitration of tyrosine containing proteins was intensified twice on day 3 of DILS overlapping with nitrating agent overaccumulation. Also, the abundance of 8-nitroguanine, a marker of nitrative modification of RNA and DNA, increased significantly on days 3 and 7 of DILS, respectively. Taken together ONOO– could be considered as a novel positive regulator of DILS fine-tuning redox environment to selective nitrative modification of biotatrgets such as tyrosine containing proteins and RNA.
This work was supported by Polish National Science Centre in the frame of the project UMO-2017/26/E/NZ4/00226
POG PP 1.19:
Nitrosative stress affects histone deacetylases and acetyltransferases in the plant pathogen Phytophthora infestans
Joanna Gajewska | Adam Mickiewicz University in Poznań, Poland, Department of Plant Ecophysiology | Poland
Phytophthora infestans (Mont.) de Bary is one of the most important plant pathogens in agriculture. The presence of a large number of acetyltransferase and deacetylase orthologs in the genome of Phytophthora species suggests that lysine acetylation of various proteins may be crucial in these fungal-like organisms. The pathogen is also able to synthesize nitric oxide, a signaling molecule that could be engaged in molecular reprogramming via changes in the histone acetylation status.
To gain insight into the acetylation status in the phytopathogen structures we performed the experiments on the avirulent (avr MP946) and virulent (vr MP977) P. infestans isolates in reference to the potato (Solanum tuberosum L.) cv. Sarpo Mira. In order to verify whether and to what extent reactive nitrogen species (RNS) affect nuclear histone deacetylases (HDACs) and acetyltransferases (HATs) at transcript and enzyme activity levels, the pathogen was treated with specific donors to mimic nitrosative stress conditions to which the pathogen is exposed during in planta growth.
The results indicated a RNS-dependent induction of HDACs activity at 2nd h after donors application; however, it reduced starting from 24h after S-nitrosoglutathione and SIN-1 (3-morpholino-sydnonimine) treatments. Among the 5 analyzed genes encoding nuclear HDACs, RNS provoked a significant increase of the transcript accumulation for HDAC1, HDAC3, and HDAC5. Analyses of HATs activity showed the strongest enzyme induction at 48h after donors treatment. Among the 13 analyzed genes encoding HATs, 6 showed altered expression via RNS. Moreover, different patterns of the gene expression were observed in both isolates.
Summing up, nitrosative stress modified HDACs and HATs at transcript and enzyme activity levels in both P. infestans isolates. Thus, the interaction of RNS-HDACs/HATs can be crucial in controlling the expression of a plethora of P. infestans genes. This research was funded by National Science Centre – project no. UMO-2018/31/B/NZ9/00355.
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Exploiting a bacterial cysteine desulfurase-sulfurtransferase fusion to develop a roGFP2-based biosensor for free cysteine
Damien Caubrière | Université de Lorraine-INRAE, UMR1136 Interactions Arbres-Microorganismes, Nancy, France
In recent years, the development of fluorescent probes has revolutionized our experimental access to physiological parameters in live cells. Genetically-encoded-probes based on redox-sensitive yellow fluorescent protein (rxYFP) and green fluorescent proteins (roGFPs), allow real-time monitoring of thiol redox dynamics, combined with the option of precise targeting to specific subcellular locations in any cellular systems or organisms including plants. For instance, the redox-sensitive GFP (roGFP2) has been fused to glutaredoxins and thiol peroxidases to allow dynamic imaging of the glutathione redox potential (EGSH) or H2O2, respectively.
Cysteine is an essential metabolite, that is required for protein synthesis but also for the production of many important sulfur-containing molecules, for the biosynthesis of cofactors such as iron-sulfur centers or molybdenum cofactors but also for the biogenesis of hydrogen sulfide. To broaden our understanding of redox biology in a cellular and physiological context, biosensors able to detect cysteine levels and its degradation/derived products are needed.
In principle, any protein domain with a catalytic cysteine undergoing a specific and reversible oxidative modification may oxidize roGFP2 unless this is sterically/structurally hampered. Cysteine desulfurases (CDs) and sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from sulfur donors to nucleophilic sulfur acceptors by forming an intermediate persulfide on a single reactive catalytic cysteine. Thus, they represent good candidates to establish genetically-encoded probes as they seem perfectly suited to transfer an efficient and selective oxidation of roGFP2. In this work, we first confirmed the ability of a natural CD-STR fusion protein to oxidize the roGFP2 in the presence of cysteine. Then, using in vitro fluorescence assays, we investigated the specificity, sensitivity and reduction (once oxidized) of a CD-STR-roGFP2 fusion. These results prompted us to express the CD-STR-roGFP2 fusion in Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana to perfom in vivo measurements.
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The Arabidopsis mitochondrial protein PPR40 modulates drought tolerance.
Kamal Kant | Biological Research Centre, Szeged | Hungary
Due to climate change, plant tolerance to environmental stresses is an important objective of breeding efforts. Combination of extreme conditions such as drought and high temperature can affect plants more than individual stresses. Our previous research revealed that the mitochondrial pentatricopeptide (PPR) domain protein 40 (PPR40), connects mitochondrial electron transport and of stress responses by influencing redox balance and ROS signaling in Arabidopsis thaliana (Zsigmond et al., 2008, Plant Physiol. 146:1721-1737). In a subsequent study influence of PPR40 on drought tolerance was characterized with T-DNA insertion mutants ppr40-1, ppr40-2 and PPR40 overexpression plants. Plant growth and survival rates as well as stress-related physiological parameters (chlorophyll fluorescence changes, relative water content, ROS accumulation and oxidative damage and proline levels) were analysed in plants subjected to drought, heat stresses and their combinations. Physiological studies were completed by complex phenotyping of the ppr40 mutants. Our results revealed that T-DNA insertion in the PPR40 gene could enhance drought tolerance by reducing water loss, stabilizing photosynthetic electron transport rates and improving viability of the mutants. Our results suggest that genes implicated in mitochondrial electron transport can be potential candidates for engineering drought tolerance in crop plants.
This research was supported by NKFI FK-128920.
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Investigating the H2O2 sensitivity of LbAP1, a YAP1-like homolog in the ectomycorrhizal fungus Laccaria bicolor.
Maarten Ottaway | Vrije Universiteit Brussel (VUB)
Ectomycorrhizal fungi (ECM) are an important group of organisms that ensure that trees can flourish in all types of environmental conditions. By providing nutrients, water and protection against stress conditions in exchange for sugars, they allow their host trees to grow. The stress response of plants has already been studied in detail. However, stress response signalling remains poorly understood in ECM fungi. In this study, we assess the potential of H2O2 to regulate a putative transcription factor, LbAP1, using a bio-informatic approach, and localisation of heterologously expressed eGFP-fused LbAP1 upon H2O2 treatment. LbAP1 is a homolog of YAP1, a key regulator of ROS responses in Saccharomyces cerevisiae. Oxidation of two cysteines in YAP1, located in the N- and C-terminal CRD, results in nuclear accumulation through masking of the NES by disulfide bond formation and transcriptional activation of target genes. In contrast to YAP1, results obtained by fluorescence microscopy showed that an LbAP1-eGFP fusion protein did not accumulate in the nucleus upon treatment of transformed Saccharomyces cerevisiae BY4741 with 0.4 mM H2O2. As C-terminal eGFP could be interfering with disulfide bond formation, the H2O2 sensitivity of eGFP-LbAP1 is currently being examined. Upon analysis of both protein sequence and structure of YAP1 and LbAP1, it was revealed that LbAP1 contains 2 Cys less than YAP1. Furthermore, the Cys responsible for nuclear localisation upon oxidation in YAP1 are not conserved in LbAP1. The NLS and NES, however, are partially conserved. Structural predictions of LbAP1 also showed that the helix structures containing these important cysteines are not conserved, possibly indicating that LbAP1 nuclear localisation is not H2O2 dependent. From these results, we can conclude that LbAP1 regulation might be H2O2 independent, and that the cellular mechanisms of ECM fungi should be investigated separately to other fungi.
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How does climate change affect Castanea sativa oxidative metabolism? – an insight into the combined effects of drought and high temperatures
Filipa Sousa | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
Chestnut (Castanea sativa Miller) is an important species throughout Europe, particularly in Portugal. However, it is highly sensitive to episodes of high temperatures and water scarcity scenarios that are increasingly common due to climate change. Given the need and importance of understanding the impact of these stress factors on the physiology of C. sativa, this study focused on the evaluation of the response of young chestnut plants (3 months old) to the combination of drought and heat (4 h/d 42 ºC). The exposure to high temperatures did not substantially affect the physiological performance of these plants, as no major impacts were found on growth and oxidative metabolism. On the other hand, after 21 days of exposure to drought, individually and in combination, this condition was the one that most affected plant growth. This effect was accompanied, in the case of individual stress exposure, by a decrease in the production of reactive oxygen species, namely superoxide anion (O2.-) and hydrogen peroxide (H2O2), as well as by an increase in lipid peroxidation and proline levels. A prevalence of the effect of drought in the co-exposure treatment was observed, although the plants subjected to the combination of stresses showed an increase in carotenoids and O2.- contents. Ongoing studies are focused on the evaluation of the photosynthetic and antioxidant response of C. sativa to these abiotic stresses ensure a holistic view on the impact of climate change on chestnut plants. Additionally, and recognizing the imperative need to develop new effective and eco-friendly approaches to overcome the harmful consequences of these unfavorable conditions, studies are also underway to assess the potential of mycorrhization as a strategy to mitigate the effects of climate change on this species.
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Mitigating the effects of climate change in tomato plants – mission (im)possible?
Bruno Sousa | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
Currently, climate change is affecting crop production worldwide. For instance, in the Mediterranean basin, comprising the most important European producers and exporters of tomato, productivity has been rapidly declining, mostly due to the increased temperatures and soil salinization, two conditions that frequently occur together in the environment. Indeed, recent studies carried out by our team showed that the combination of these two stressors affects tomato plants more severely than the sum of the individual effects. In this sense, it becomes highly important to develop and establish sustainable strategies to improve crop growth under this scenario of climate instability. Thus, the goal of this study was to assess how the exogenous application of two phytohormones [brassinosteroids (BRs; 1 µM 24-epibrassinolide) and strigolactones (SLs; 5 µM GR24)] and a beneficial element (silicon – Si; 2 mM) can modulate the response of tomato plants (Solanum lycopersicum L. var. cerasiforme) to heat and salinity. After 7 d of acclimation to growth chamber conditions, salt stress was applied through NaCl irrigation (100 mM) every alternate day during 28 d, with heat stress being applied in the last 21 d, through exposure to 42 ºC for 4 h every other day. SLs, BRs, and Si treatments were given individually as a foliar spray, twice per week, throughout the experiment. After the growth period, the obtained data indicated that regardless of the applied compound, the macroscopic effects observed in the stress situation were maintained, as well as those related to the oxidative (hydrogen peroxide and lipid peroxidation) and photosynthetic (chlorophyll and carotenoids) metabolism. However, it is important to highlight that there was a small tendency for higher biomass in all sprayed plants, as well as increased glutathione and ascorbate, and reduced proline levels in the case of plants treated with SLs, suggesting the activation of protective pathways.
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Ameliorative effects of ascorbate on increasing tomato tolerance to NiO nanomaterials - an in vitro approach
Sofia Spormann | GreenUPorto - Sustainable Agrifood Production Research Center & INOV4AGRO | Portugal
While nanomaterials (NMs) offer wide-ranging solutions, their intensified use has been resulting in the contamination of the environment, posing ecotoxicological risks to diverse organisms, including plants. In this sense, it becomes important not only to understand the phytotoxicity of NMs, but also to find efficient and sustainable strategies to enhance plant tolerance to these emergent contaminants. Thus, this study aimed at assessing the potential of ascorbic acid (AsA), a powerful antioxidant (AOX), in enhancing the tolerance of in vitro grown tomato seedlings to nickel oxide NMs (nano-NiO). For this purpose, seeds of Solanum lycopersicum L. cv. Micro-Tom were germinated in half-strength MS medium supplemented with 30 mg L-1 nano-NiO alone or in combination with 150 mg L-1 AsA. A control situation, without nano-NiO nor AsA, was also included. After 28 days of exposure, the growth and development of S. lycopersicum was severely repressed by nano-NiO, with evident phytotoxicity symptoms (leaf chlorosis and necrosis), that did not translate, however, into severe redox disorders. Even so, proline levels, SOD and DHAR activities were diminished in shoots of nano-NiO-exposed plants, while glutathione, phenols, CAT and GR activities were increased. In response to the co-exposure to AsA, nano-NiO-induced growth inhibition was efficiently counteracted, being accompanied by a more notorious response of the AOX network, especially of glutathione, phenolics, SOD and GR. Surprisingly, the solo AsA administration in the culture medium suppressed growth and development of tomato seedlings, increased the lipid peroxidation of membranes and inhibited key enzymes of the AsA-glutathione cycle, in spite of enhancing the total AOX capacity, with increased levels of proline, phenols and glutathione. Overall, exposure to nano-NiO negatively impacted tomato seedlings’ growth, and co-application of exogenous AsA had stress-ameliorative effects by enhancing the AOX response to counteract nano-NiO-induced growth inhibitory effects.
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Analysis of the different isoforms of Arabidopsis monodehydroascorbate reductase
Dongdong XU | Université Paris-Saclay, Institut of Plant Sciences (IPS2) | France
Hydrogen peroxide (H2O2) is a major reactive oxygen species (ROS) whose level must be carefully controlled to allow optimal cellular signaling. One important plant antioxidant enzyme that removes excess H2O2 is ascorbate peroxidase (APX), which reduces this reactive molecule to water. Ascorbate peroxidase requires ascorbate and, in reducing H2O2, produces monodehydroascorbate (MDHA), which must be regenerated to ascorbate by reductases.
One of the regenerating enzymes is MDHA reductase, which converts MDHA directly to ascorbate.
In Arabidopsis, several MDHAR isoforms exist and they are found in different subcellular compartments. While there are two peroxisomal isoforms (MDHAR1 and MDHAR4), MDHAR2 and MDHAR3 are found in the cytosol, and dual targeting of MDHAR5/6 allows expression in mitochondria and chloroplasts, respectively. It is widely thought that MDHAR plays an important role in the response of plants to oxidative stress by maintaining the redox state of intracellular ascorbate, but few specific detailed studies of the different isoforms have been conducted.
We are performing a detailed comparison of the different MDHAR isoforms. To establish how these enzymes work within the cellular redox network, a comparison of their affinities for NADH and NADPH is being conducted using recombinant enzymes while a reverse genetics approach using specific mutants aims at assigning specific biological functions to each gene. To this end, single and double mutant lines are being produced. Questions we aim to answer include whether the different MDHARs show specificity in biochemical properties as well as the importance of each for the functioning of plants under optimal and stress conditions.
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The Root-knot nematode effector Mj-NEROSS suppresses plant immunity by interfering with the ROS production in plastids
Yujin Chen | Faculty of Bioscience Engineering -UGent | Belgium
Plant-parasitic nematodes produce effectors to overcome plant immunity and finetune plant cellular processes. Molecular mechanisms of how effector proteins co-opt plant processes, especially plant immunity to support nematode survival, have been intensively investigated, but they are still poorly understood. Identifying protein-protein interactions is crucial for understanding this cross-kingdom network.
Using the high throughput screening technique Y2H-seq, we have identified tomato proteins involved in various cellular processes interacting with M. javanica effectors. Among those, Mj-NEROSS (Nematodes effector involved in ROS suppression, previously referred to as 4D01 or Msp3) was found to interact with a Heavy metal transport/detoxification superfamily protein and a Rieske iron-sulphur protein (ISP), one of the putative subunits of the cytochrome b6f complex. Furthermore, we confirmed direct interaction in planta and showed that ISP is a conserved dicot target of Mj-NEROSS.
We showed that the plastid localization of Mj-NEROSS plays a crucial role in its interaction with ISP. Once this interaction has been established, a significant decrease in electron transport rate and subsequently in the host's reactive oxygen species production is observed. Furthermore, we reveal that the presence of the effector in the plastids leads to changes in genes expression. Importantly, we show that the differentially expressed genes (DEGs) are involved in ROS production, protein folding recognition, and upregulation of oxidative phosphorylation.
We hypothesized that DEGs are most likely subsequent consequences of interaction of Mj-NEROSS with ISP and biochemical communication between plastids and the nucleus, leading to suppression of plant basal defense and attenuation of host resistance to the nematode infection.
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MPK4, CIA2 and CIA2-LIKE are required for optimal photosynthesis and oxidative stress responses in plants
Prof. Dr. Stanisław Karpiński | Warsaw University of Life Sciences - SGGW | Poland
Our functional analysis of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) for hybrid aspen (Populus tremula × tremuloides) grown under natural field conditions for several seasons provided evidence of the role of MPK4 in the genetic and environmental regulation of stomatal formation, differentiation, signalling, and function; control of the photosynthetic and thermal status of leaves; and growth and acclimation responses. Divergence between absorbed energy and assimilated energy is a bottleneck, and MPK4 can participate in the control of energy dissipation (thermal effects). Furthermore, MPK4 can participate in balancing the photosynthetic energy distribution via its effective use in growth or redirection to acclimation/defence responses.
The vast majority of chloroplast proteins are nuclear-encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses (e.g. SAA and SAR) that influence nuclear gene expression. Signalling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual-localized TFs involved in chloroplast retrograde signalling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homologue CIA2-like (CIL) are involved in the regulation of Arabidopsis responses to UV-AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signalling events in the chloroplast-nucleus cross-talk.
1. Witoń et al., Plant Phys. 2021, 186: 2190-2204; 2. Gawroński et al., Plant J. 2021 105: 619–638.
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DPI-dependent production of superoxide in reproductive tissues of the olive tree (Olea europaea L.)
Dr. Juan de Dios Alché | Estación Experimental del Zaidín (CSIC) | Spain
Reactive Oxygen Species (ROS) are compounds derived from oxygen with important implications in biological processes in plants, some of them related to reproduction. Among ROS, superoxide is the primary oxidant, since an array of other ROS are eventually derived from this anion. There-fore, analysis of the molecular systems able to generate this molecule and the cellular compart-mentalization of these events is of paramount importance. We have used the fluorochrome DCFH2-DA and the chromogenic substrate NBT in association with DPI (a specific inhibitor of Rboh enzymes generating superoxide in plants) in combination with confocal microscopy and stereomicroscopy, respectively to identify cell localization of ROS in general, and superoxide ac-cumulation in olive reproductive tissues. A significant production of both ROS and superoxide has been described, showing a fairly precise spatial and temporal location throughout olive flower development. The reduction of the NBT signal after the addition of DPI suggests that the generation of superoxide is largely due to Rboh or other flavin oxidase activity. At the subcellular level, accumulation of O2.- has been located in the plasma membrane of mature pollen and germi-nated pollen, as well as in the rough endoplasmic reticulum and in mitochondria.
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).
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The role of NADPH oxidases-dependent ROS in regulating leaf gas exchange and ion flux in Arabidopsis plants under Cd stress
Prof. María C. Romero Puertas | Estación Experimental del Zaidín-CSIC | Spain
The involvement of NADPH oxidases in the regulation of K and Cd flux and photosynthesis was assessed in cadmium-challenged wild type and three RBOHs (Respiratory Burst Oxidase Homologues) mutants of Arabidopsis plants (AtrbohC, AtrbohD, and AtrbohF) using different approaches. Plants were grown under hydroponic conditions supplemented or not with 50 µM for 24 h. Results showed that Cd differentially affect photosynthesis and trasnpiration in WT and Atrbohs mutants. Electrophysiological experiments revealed that knocking out RBOHs induced a more Cd2+ influx compared to WT with differential trend in mature and elongation zone, being observed in the Atrboh mutants. Under Cd stress the three Atrbohs mutants had a more ability to retain K+ in the elongation root zone compared to WT, while in leaves, a dramatic reduction in K+ influx was observed in Atrbohs mutants and a change from efflux to intensive influx was registered in WT. The analysis of expression of several K+ and Cd2+ genes transporters and transcription factors suggest that RBOH-dependent H2O2 regulates ion homeostasis and Cd in a highly complex process involving multilevel regulation from transpirational water flow to transcriptional and posttranslational modifications of K/metals transporters.
This work was supported by FEDER Funds (A1123060E00013), Junta deAndalucía I+D+I grants Ref PY20_00364, and Spanish Ministry of Science and Innovation Ref PGC2018-098372-B-I00.