Where does the EU-path on new genomic techniques lead us?

Recently, the European Commission (EC) published a regulatory proposal on plants generated with new genomic techniques (NGTs) (5 July 2023). According to this proposal, NGT plant applications are categorized into category 1 NGT (NGT1) and category 2 NGT (NGT2) based on their molecular characteristics, which diverges from the current legislation centered around Directive 2001/18/EC. To demonstrate where the path of the proposal leads to in practice, we applied the proposed criteria for categorization to a list of NGT plant applications currently in the commercialization pipeline. Combining literature research and a descriptive statistical approach, we can show that 94% of the plant applications affected by the EC proposal, would be classified as NGT1 and thus would receive market approval without risk assessment, monitoring, and sufficient labeling provisions. The remaining 6% of applications would be classified as NGT2 plants, for which, in deviation from the current regulation, an adapted risk assessment is proposed. Screening of the intended traits in the pipeline highlights that certain NGT1 plants can pose similar environmental risks (e.g., invasiveness) to other genetically modified organisms (GMOs), as defined in Directive 2001/18/EC. For example, NGT1 applications based on RNA interference technology can exhibit insecticidal effects with potential side effects on non-target organisms (i.e., other insects). Our quantitative and case-specific elaboration of how the current EC regulatory proposal would affect the environment, health, and consumer protection will be informative for decision-makers and politicians.

Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

Quantification of Redox-Sensitive GFP Cysteine Redox State via Gel-Based Read-Out.

To date, fluorescent protein biosensors are widely used in research. In vivo, they can be applied to dynamically monitor several physiological parameters in various subcellular compartments. Redox-sensitive green fluorescent protein 2 (roGFP2) senses the glutathione redox potential via a disulfide bridge formed between neighboring beta-strands of its beta-barrel structure. As changes in redox state affect both excitation maxima of roGFP2 oppositely, sensor responses are ratiometric. The reaction mechanism of roGFP2 is well characterized and involves an intermediate S-glutathionylation step. Thus, roGFP2 is also used in enzymatic in vitro assays, e.g., assessing glutaredoxin kinetics. In addition to the fluorescent read-out, the roGFP2 redox state can also be determined by differential migration on a non-reducing SDS-PAGE. This read-out mode may be beneficial in some applications, e.g., if mass-spectrometric analysis of posttranslational cysteine modifications is desired. Here, we describe a protocol for gel-based fluorescent read-out of the roGFP2 redox state, as well as modification of free cysteines by maleimide-based reagents.

Reductive stress triggers ANAC017-mediated retrograde signaling to safeguard the endoplasmic reticulum by boosting mitochondrial respiratory capacity.

Redox processes are at the heart of universal life processes, such as metabolism, signaling, or folding of secreted proteins. Redox landscapes differ between cell compartments and are strictly controlled to tolerate changing conditions and to avoid cell dysfunction. While a sophisticated antioxidant network counteracts oxidative stress, our understanding of reductive stress responses remains fragmentary. Here, we observed root growth impairment in Arabidopsis thaliana mutants of mitochondrial alternative oxidase 1a (aox1a) in response to the model thiol reductant dithiothreitol (DTT). Mutants of mitochondrial uncoupling protein 1 (ucp1) displayed a similar phenotype indicating that impaired respiratory flexibility led to hypersensitivity. Endoplasmic reticulum (ER) stress was enhanced in the mitochondrial mutants and limiting ER oxidoreductin capacity in the aox1a background led to synergistic root growth impairment by DTT, indicating that mitochondrial respiration alleviates reductive ER stress. The observations that DTT triggered nicotinamide adenine dinucleotide (NAD) reduction in vivo and that the presence of thiols led to electron transport chain activity in isolated mitochondria offer a biochemical framework of mitochondrion-mediated alleviation of thiol-mediated reductive stress. Ablation of transcription factor Arabidopsis NAC domain-containing protein17 (ANAC017) impaired the induction of AOX1a expression by DTT and led to DTT hypersensitivity, revealing that reductive stress tolerance is achieved by adjusting mitochondrial respiratory capacity via retrograde signaling. Our data reveal an unexpected role for mitochondrial respiratory flexibility and retrograde signaling in reductive stress tolerance involving inter-organelle redox crosstalk.

Plasticity in plastid redox networks: evolution of glutathione-dependent redox cascades and glutathionylation sites.

BACKGROUND: Flexibility of plant metabolism is supported by redox regulation of enzymes via posttranslational modification of cysteine residues, especially in plastids. Here, the redox states of cysteine residues are partly coupled to the thioredoxin system and partly to the glutathione pool for reduction. Moreover, several plastid enzymes involved in reactive oxygen species (ROS) scavenging and damage repair draw electrons from glutathione. In addition, cysteine residues can be post-translationally modified by forming a mixed disulfide with glutathione (S-glutathionylation), which protects thiol groups from further oxidation and can influence protein activity. However, the evolution of the plastid glutathione-dependent redox network in land plants and the conservation of cysteine residues undergoing S-glutathionylation is largely unclear. RESULTS: We analysed the genomes of nine representative model species from streptophyte algae to angiosperms and found that the antioxidant enzymes and redox proteins belonging to the plastid glutathione-dependent redox network are largely conserved, except for lambda- and the closely related iota-glutathione S-transferases. Focussing on glutathione-dependent redox modifications, we screened the literature for target thiols of S-glutathionylation, and found that 151 plastid proteins have been identified as glutathionylation targets, while the exact cysteine residue is only known for 17% (26 proteins), with one or multiple sites per protein, resulting in 37 known S-glutathionylation sites for plastids. However, 38% (14) of the known sites were completely conserved in model species from green algae to flowering plants, with 22% (8) on non-catalytic cysteines. Variable conservation of the remaining sites indicates independent gains and losses of cysteines at the same position during land plant evolution. CONCLUSIONS: We conclude that the glutathione-dependent redox network in plastids is highly conserved in streptophytes with some variability in scavenging and damage repair enzymes. Our analysis of cysteine conservation suggests that S-glutathionylation in plastids plays an important and yet under-investigated role in redox regulation and stress response.