Spoel, Steven

Le Bihan, Thierry

Kneeshaw, Sophie

Biotechnology and Biological Sciences Research Council (BBSRC)

2016-12-07T15:09:57Z

2016-12-07T15:09:57Z

2016-11-29

Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches.The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.

http://hdl.handle.net/1842/18754

en

The University of Edinburgh

Kneeshaw, S., Gelineau, S.,Tada, Y., Loake, G. J., and Spoel, S. H. (2014). Selective protein denitrosylation activity of Thioredoxin-h5 modulates plant immunity.Molecular-Cell,56(1),153–62.

2100-12-31

redox-based PTMs

transcriptional regulators

Thioredoxins

TRXh5

oxidative stress

Molecular mechanisms of redoxin-mediated signalling in plant immunity

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

Restricted Access