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|Title: ||SUPPRESSOR OF GAMMA RESPONSE 1 and glutathione: Key regulators of cadmium-induced stress responses in Arabidopsis thaliana|
|Authors: ||Hendrix, Sophie|
|Advisors: ||Cuypers, Ann|
|Issue Date: ||2019|
|Abstract: ||Pollution of soils and water with cadmium (Cd) as a consequence of anthropogenic activities poses a major threat to human health, mainly via Cd uptake in plants and its accumulation into the food chain. Cadmium disturbs multiple physiological processes in plants and thereby inhibits plant growth. At the cellular level, Cd induces an oxidative challenge, characterized by the increased production of reactive oxygen species (ROS). Despite their important roles in signal transduction, ROS can cause damage to cellular macromolecules such as DNA, proteins and lipids when present in increased concentrations. To prevent oxidative damage, plant cells possess an extensive antioxidative defense system comprising enzymatic compounds such as superoxide dismutases (SODs) and non-enzymatic compounds including glutathione (GSH). In addition to its function in antioxidative defense, GSH is also involved in Cd chelation, both directly and through its role as a precursor for the biosynthesis of metal-chelating phytochelatins (PCs). Moreover, previous research demonstrated that GSH is involved in cell cycle regulation in roots. The classical plant cell cycle consists of four phases, including DNA replication and cell division, and is regulated by the coordinated activity of cyclin-dependent kinases (CDKs) complexed with regulatory cyclins. During stress conditions, cell cycle progression is often affected though the DNA damage response (DDR). Upon perceiving DNA damage, this pathway is activated and regulates the cell cycle, DNA repair and programmed cell death. A central player in the plant DDR is the transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), which is considered as a functional homologue of p53 in animals. The main aim of this study was to investigate the role of SOG1 and GSH in Cd-exposed Arabidopsis thaliana plants, focusing on their involvement in the DDR and the Cd-induced oxidative stress response.
As information regarding the effects of Cd exposure on cell cycle regulation in plants was mostly limited to roots and cell cultures, the first part of this study aimed to investigate Cd-induced effects on the cell cycle in A. thaliana leaves (Chapter 3). To this end, the extent of cell division and endoreduplication were determined in plants exposed to 5 μM Cd for 72 h or 12 days. Cadmium exposure was shown to inhibit both cell cycle variants and this effect accumulated over time. Furthermore, expression levels of three CDK inhibitors of the SIAMESE-RELATED (SMR) family and other SOG1-regulated genes were significantly increased upon Cd exposure, indicating that Cd activates the DDR at the transcript level.
To further unravel the involvement of SOG1, SMR4, SMR5 and SMR7 in Cd-induced stress responses, multiple parameters related to the DDR and oxidative stress were compared between leaves of wild-type (WT) A. thaliana plants, sog1-7 mutants and smr4/5/7 triple mutants (Chapter 4). In general, Cd-induced responses were highly similar between leaves of the WT and the smr4/5/7 mutant, indicating either that these SMRs are not involved in the Cd-induced DDR and oxidative stress response or that bypass mechanisms compensate for the lack of functional SMRs in the mutant. In contrast, the sog1-7 mutant exhibited a lower Cd sensitivity in comparison to WT plants upon acute Cd exposure. The Cd-induced upregulation of many DDR-related genes was less pronounced or even completely absent in sog1-7 mutant leaves. Furthermore, the inhibition of cell division and endoreduplication observed in WT plants was also largely absent in the mutant genotype, demonstrating the role of SOG1 in the Cd-induced DDR. Interestingly, Cd-induced increases in leaf hydrogen peroxide and GSH concentrations were delayed in the sog1-7 mutant. This response coincided with a lack of transcriptional activation of genes involved in Cd-induced oxidative signaling, which might be explained by the fact that OXIDATIVE SIGNAL-INDUCIBLE 1 (OXI1) – an important player in the oxidative signaling pathway – is a direct target gene of SOG1. Upon prolonged exposure, however, a stronger Cd-induced inhibition of reproductive growth was observed in the mutant, which might be related to a higher extent of oxidative damage, as the level of Cd-induced lipid peroxidation in sog1-7 mutant leaves significantly exceeded that in WT leaves. These data indicate that, besides its involvement in the Cd-induced DDR, SOG1 plays an additional role in the oxidative stress response.As GSH was previously shown to influence cell cycle regulation and is known to play an important role in Cd-induced stress responses, the next part of this study aimed to elucidate the involvement of GSH in cell cycle regulation in Cd-exposed A. thaliana (Chapter 5). To this end, the extent of cell division and endoreduplication were compared between leaves of Cd-exposed WT plants and the partially GSH-deficient cadmium-sensitive 2-1 (cad2-1) mutant. The obtained results strongly support a role for GSH in these processes in Cd-exposed plants, as negative effects of Cd on both cell division and endoreduplication were significantly more pronounced in leaves of cad2-1 mutants as compared to WT plants. In addition, the Cd-induced increased extent of endoreduplication observed in young leaves shortly after their emergence, was absent in the cad2-1 mutant. Furthermore, the Cd-induced transcriptional activation of the DDR was not observed in leaves of the cad2-1 mutant, suggesting that GSH is required for SOG1 activation.
Finally, this study aimed to further unravel the role of GSH in the Cd-induced oxidative stress response, specifically focusing on the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7 (SPL7)-mediated copper (Cu) deficiency-like response (Chapter 6). This response is characterized by the opposite regulation of Cu/zinc SODs (CSD1/2 downregulation) and iron SOD 1 (FSD1 upregulation). Our results showed that this SPL7-mediated response was delayed in roots of the cad2-1 mutant. To reveal similarly regulated mechanisms, a whole transcriptome analysis was performed in WT and cad2-1 mutant plants exposed to Cd. The results indicated that ethylene-related genes were more strongly upregulated upon Cd exposure in roots of mutant as compared to WT plants, whereas the opposite was observed in leaves. A highly similar response was seen for genes related to endoplasmic reticulum (ER) stress. The latter is a consequence of the accumulation of unfolded or misfolded proteins in the ER during stress conditions. As the ER hosts both SPL7 and the ethylene receptor ETR1, we propose a central role for the ER in the Cd-induced stress response through integrating GSH-related responses, ethylene signaling and the SPL7-mediated Cu deficiency-like response.|
|Type: ||Theses and Dissertations|
|Appears in Collections: ||PhD theses|
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