Thioredoxins enable selective and reversible redox signalling in plants
Item statusRestricted Access
Embargo end date20/01/2024
Bleau, Jade R.
Accumulation of reactive oxygen species (ROS) in eukaryotic cells is associated with several biological processes, including environmental stress responses. ROS levels are usually maintained at low levels by an extensive network of antioxidants, as dysregulation of ROS can cause severe cellular damage. However, ROS also play important signalling roles in large part through the oxidation of reactive cysteine residues in key regulatory proteins. These oxidative post-translational modifications (oxPTMs) are dynamic and diverse, generating a highly adaptable and signal-responsive proteome. While some oxPTMs are undesirable, the majority are reversible and thus, can be utilised as molecular signalling switches. Reversibility of oxPTMs can largely be attributed to a superfamily of oxidoreductases, known as Thioredoxins (TRXs). Plants contain a large number of diverse TRXs which are localised throughout the cell, with several sharing the same subcellular localisation. While TRXs are known to be involved in many biological processes, the extent to which they signal selectively rather than redundantly remains unclear. The work in this thesis explores the versatility and selectivity of TRX-mediated redox signalling in response to stress. In Arabidopsis, the oxidoreductase, Nucleoredoxin1 (NRX1), protects the cell in ROS-rich environments by guarding antioxidant enzymes involved in H2O2 scavenging. However, little is known about the role NRX1 plays in curbing oxidative stress independent of antioxidant enzymes. Chapter 3 shows that NRX1 selectively rescues oxidative stress responses in antioxidant-deficient plants. While expression of NRX1 was unable to rescue pad2 plants that are deficient in the antioxidant glutathione from oxidative cell death, it rescued catalase-deficient (cat2) plants from cell death induced by high light and the oxidative stressor methyl viologen (MV). The data in this chapter suggests that NRX1 rescues catalase mutants from light-induced oxidative stress by partially restoring ROS-induced changes in gene expression back to near wild-type levels. Interestingly, NRX1 rescued expression of TRXh5, a stress-induced cytosolic TRX member with major roles in biotic and abiotic stress resistance. In Chapter 4 it is shown that selective rescue of stress responses extends to other members of the TRX-h family. TRX-h members showed a distinct separation in their ability to rescue differentially induced stress responses. While both cat2 and pad2 mutants are immune compromised, TRX-h members display strong preference for rescuing immunity only in pad2 mutants. Additionally, different TRX-h members selectively rescued MV-induced cell death in either pad2 or cat2 mutants. Taken together, these results suggest that TRXs each have specific substrate repertoires that control different ROS signalling pathways in response to stress. Whilst PTMs allow for increased regulation of proteins, there is still much to uncover about how PTMs themselves are regulated. Chapter 5 shows that NRX1 may control oxPTMs of accessory proteins of the ubiquitin-proteasome system (UPS). The UPS is a sophisticated pathway that coordinates the degradation of intracellular proteins by labelling unstable or damaged proteins with chains of the small post-translational modifier, ubiquitin, thereby targeting them for proteasomal degradation. The work in Chapter 5 reports that NRX1 maintains the activity of deubiquitinase (DUB) enzymes responsible for cleaving ubiquitin chains. It is shown that the DUB, UBP13, is subject to H2O2-induced oxidation, which inhibited its DUB activity. Furthermore, NRX1 physically interacted with UBP13 and acted as a molecular reductant to activate and maintain UBP13 activity. Preliminary findings indicate that NRX1 may regulate UBP13-mediated hormonal signalling, as JA hypersensitivity observed in transgenic plants overexpressing UBP13 was impaired in the absence of NRX1. Overall, the work in this thesis demonstrates that TRX family members are highly selective regulators of diverse ROS signalling pathways, enabling plants to tailor their responses depending on the stresses they encounter. It further shows that cross-talk between different PTMs (i.e. oxPTMs and ubiquitin) enables precise regulation of the proteome during oxidative stress and hormonal signalling.