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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.

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.

Abstract 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.