Structural and functional characterisation of chloroplast signaling
Creators: name: Chan, Kai Xun
Publisher: name: The Australian National University
Year: 2015

description: The evolution of multiple cellular compartments in higher eukaryotes has necessitated additional complexity in intracellular signaling networks to coordinate cellular function. In the model plant Arabidopsis thaliana (Arabidopsis), a number of metabolite and mobile transcription factor signals involved in organelle-nucleus signaling in response to perturbations in chloroplasts and mitochondria, where Reactive Oxygen Species (ROS) are produced, have been elucidated. One such signal is the sulfation byproduct 3'-phosphoadenosine 5'-phosphate (PAP), which is degraded in the chloroplasts and mitochondria. PAP accumulates during drought and high light stress, is capable of moving between subcellular compartments, and inhibits the activity of 5'- 3' Exoribonucleases (XRNs) to alter expression of stress-responsive genes. Despite these advances in identifying organelle-nucleus signals and characterizing their downstream effects, the molecular basis for the generation of retrograde signals such as PAP remain enigmatic. Understanding how the chloroplast and mitochondria regulate the accumulation of PAP during oxidative stress will provide insights into mechanisms for the integration of stress perception and signal initiation within plant energy systems. This thesis therefore undertakes the challenge of understanding the regulation of PAP accumulation during oxidative stress by interrogating three key aspects of PAP metabolism within secondary sulfur metabolism: biosynthesis of PAP and its precursor 3'-phosphoadenosine 5'-phosphosulfate (PAPS), transport of these metabolites between different subcellular compartments, and the catabolism of PAP. Genetic and functional characterization of PAP biosynthesis, targeting four Adenosine Phosphosulfate Kinase (APK) isoforms which are rate-limiting for PAPS and PAP production, indicate absence of coordinated up-regulation of PAP biosynthesis for generation of this retrograde signal during drought stress. That said, some transcriptional evidence also suggests possible compartment-specific up-regulation of PAPS biosynthesis and involvement of specific Sulfotransferase (SOT) enzymes in producing PAP. This thesis additionally presents genetic evidence for redundancy of PAPS/PAP transport between subcellular compartments during growth and oxidative stress, and outlines new strategies to manipulate PAPS/PAP transport to achieve physiological outcomes such as stomatal closure. The major finding of this thesis concerns the regulation of PAP catabolism. Characterization of the key PAP catabolic enzyme SAL1 at the biochemical and structural levels reveals that SAL1 is redox-regulated via formation of an intramolecular disulfide, and is additionally sensitive to glutathionylation. Intriguingly, dimerization is a prerequisite for formation of the intramolecular disulfide, but not for glutathionylation, highlighting the multiple pathways for redox regulation of SAL1. Disulfide formation, dimerization and/or glutathionylation significantly down-regulate SAL1 catabolic activity against PAP in vitro and in vivo. These post-translational mechanisms collectively exert a strong structural rigidification of the enzyme that impairs its ability to degrade PAP, thus allowing PAP to accumulate and activate this retrograde signaling pathway. This thesis thus posits that the redox regulation of SAL1 allows it to function as a reversible, single-component sensor of oxidative stress within organelles and as a switch for retrograde signaling. The final section of this thesis attempts to explore the role and regulation of SAL1 beyond the plant kingdom. All components (regulator, signal, target) of the SALl:PAP:XRNs pathway are conserved across evolutionary scale. Do SAL1 homologues across kingdoms therefore also function as stress sensors/signal activators, and are they regulated in the same way? Structural, biochemical and in vivo characterization of SAL1 homologues in bacteria, yeast and humans reveal that the redox regulation is conserved across the three main eukaryotic kingdoms. Hence, SALTPAP are not just components of sulfur metabolism but may have taken up "moonlighting" roles in oxidative stress signaling across evolution. This thesis has highlighted how interrogation of plant secondary metabolism may provide insights into organelle signaling. The key finding of how redox regulation of SAL1 allows integration of ROS and oxidative stress perception with the generation of a stress signal provides the foundations for further investigation into what is an embryonic stage of stress signaling cascades that largely remains enigmatic.