POG OP 4.1:
S-nitrosylation regulates immunity against infection across life kingdoms
Prof. Gary Loake | University of Edinburgh | United Kingdom
A key feature of innate immunity in both plants and animals is the rapid engagement of a nitrosative burst leading to the accumulation of the small, redox-active signalling molecule, nitric oxide (NO). The emerging data suggests that subsequently NO has a central role in orchestrating the immune response through the reprogramming of both gene expression and protein function. The predominant route for NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein Cysteine thiol (S-H) group to form an S-nitrosothiol (S-NO). This redox-based, post-translational modification can regulate protein function in an analogous fashion to other more well-established post-translational modifications, for example, phosphorylation. Thus, S-nitrosylation can regulate enzyme activity, protein localisation, DNA binding, protein-protein interactions and metabolite binding, among others.
We are employing a number of diverse genetic and molecular strategies to further uncover the role of NO and cognate S-nitrosylation in the establishment of plant immunity in both model and crop plants. More recently, we have also begun to explore the role of this redox control mechanism in a model animal system. We will report some of our latest findings.
POG OP 4.2:
Characterization of mitochondrial electron transport mutants under stress conditions
Dr. Laura Zsigmond | Biological Research Centre, Szeged | Hungary
Plants responses to adversely altered environmental conditions encompass changes in plant growth and development as well as in photosynthesis, respiration, metabolite assimilation and catabolism. Accumulation of reactive oxygen species (ROS) is a consequence of abiotic stresses when ROS can damage proteins, lipids, and other macromolecules and therefore create additional oxidative stress for the plants. Although chloroplasts are main source of ROS, mitochondria are also important in the maintenance of the cellular redox homeostasis and regulation of cellular ATP supply in adaptive responses to stress conditions. In mitochondria, over-reduction of the electron transport chain is the primary reason for ROS accumulation, which can be reduced by protecting and stabilizing the electron flow.
To reveal the importance of genes encoding the mitochondrial proteins in stress responses, we analyzed insertion mutants of 12 Arabidopsis thaliana genes, encoding the subunits of Complex I and III of mitochondrial electron transport. Phenotypes of the mutants were characterized in osmotic, salt and oxidative stress conditions. Morphological alterations and differences in tolerance to drought and salinity were revealed through germination and growth tests and by complex phenotyping. One mutant was characterized in detail in which the mutation disrupted the NDUSF8.2 gene. The ndusf8.2-1 mutant was hypersensitive to oxidative stress, although had less hydrogen peroxide level and lower lipid peroxidation rates under osmotic stress. Changes in chlorophyll fluorescence under stress treatments suggested that this Complex I mutation can influence photosynthesis as well. Moreover, alteration were found in activities of mitochondrial complexes, in ATP/NAD(P)H levels and activities and expression of the alternative oxidoreductases (altNDs, AOXs). Our data revealed that NDUSF8.2 is important in plants stress responses, and strong correlation exist between mitochondrial functions, photosynthetic activity and energy production.
This research was supported by NKFI FK-128920.
POG OP 4.3:
ANAC089 transcription factor is an ABA and redox molecular player during seed germination and stress
Dr. Pablo Albertos | University of Salamanca | Spain
Seed dormancy and germination are complex traits regulated by the interaction of a plethora of signaling molecules, including phytohormones (abscisic acid, ABA) and plant growth regulators (nitric oxide, NO) (Sanz e tal., 2015). A genetic screening in 3μM (+)-S-ABA coupled to the effect of the NO scavenger (carboxy-2-phenyl-4,4,5,5- tetramethylimidazolin-1-oxyl-3-oxide, cPTIO) was performed. We have previously described the identification of two mutants gap1 and gap2 (germination in ABA and cPTIO1 and 2) showing ABA- and cPTIO-insensitive phenotypes in the transition from dormancy to germination and the characterization and positional cloning of one of them, gap2/abi5 mutant (Albertos et al., 2015). Concerning the other locus, we found that GAP1 encodes ANAC089 transcription factor, a member of the NAC (NAM-ATAF1,2-CUC2) family with a critical membrane-bound domain and extra-nuclear location. We demonstrated that mutants lacking the membrane-related domain of ANAC089 displayed ABA, salt, osmotic and cold stresses insensitivity revealing a repressor function of ABA and abiotic stresses responses. In addition, these ANAC089 truncated mutants exhibited higher endogenous NO levels avoiding the effect of NO-depletion during seed germination. Consistently, translocation of ANAC089 protein to the nucleus was directed by changes in cell redox status after NO- and redox-related compound treatments. Whole-genome transcriptional profiling uncovered the existence of different groups of ABA- and redox-related genes that are differentially regulated by ANAC089. The DNA binding specificity of the ANAC089 TF following a microarray-based approach is also fully provided demonstrating that can specifically bind to the core cis-regulatory element GCGTCAGC present in the promoters of ANAC089 regulated genes. Collectively, our results indicate that ANAC089 transcription factor integrates ABA signaling with NO levels to modulate redox homeostasis as a novel master regulator during seed germination and stresses in Arabidopsis (Albertos et al., 2021).
Funding: P. A. María Zambrano Grant, Next Generation EU. O. L. PID2020-119731RB-I00, Junta CyL SA137P20, CLU-2018-04.
POG OP 4.4:
Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics
Dr. José Manuel Ugalde | University of Bonn | Germany
Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress induced by dithiothreitol, was delayed in ero1 ero2. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.
POG OP 4.5:
Cellular responses of maize roots to long-term cadmium exposure: Alterations in class III peroxidases, tonoplast and plasma membrane sub-proteomes
Sabine Lüthje | Universität Hamburg | Germany
Abiotic stress has a considerable influence on important processes in plants. Cadmium (Cd) naturally occurs in volcanic soils and is accumulated in the environment by anthropogenic activities. Its uptake by plants and animals cause contaminated food products and thereby health problems in humans. Although Cd is not a redox active element it induces a concentration dependent oxidative stress in organisms via indirect mechanisms and replacement of bivalent cations in cell walls, chlorophyll or proteins. The mechanisms that counteracts this oxidative stress in plants is not completely understood and needs further elucidation. The plasma membrane is one of the first targets of the stressor, involved in Cd uptake, cell wall modification and antioxidative response. For detoxification Cd has to pass the tonoplast, to be stored inside the vacuole. Soluble and membrane-bound class III peroxidases have functions in ROS scavenging, membrane protection and cell wall modification.
In the present study changes in peroxidase activity were investigated for maize (Zea mays L.) roots after 18 days of Cd exposure. The guaiacol peroxidase activity was significantly higher in stressed samples compared to controls. Increases of activity were found for the soluble fraction and at the plasma membrane. Substrate specificity of the partially purified soluble class III peroxidases indicated a preference for ferulic acid > coniferyl alcohol > esculetin > scopoletin for cadmium induced isoenzymes. Plasma membrane-bound class III peroxidases were separated by 2D-PAGE (native IEF/non-reducing SDS-PAGE) revealing ZmPrx85 as putative marker for Cd stress. Although function of ZmPrx85 will need further proofs, strong evidence was given for a role in membrane protection and cell wall reinforcement. The detailed analyses of sub-proteome fractions from plasma membrane and tonoplast demonstrated significant changes in the abundances of signalling, transport and membrane trafficking compounds. Besides, alterations were observed for proteins involved in cell death, protein homeostasis and modification