Glutathione as key regulator of early responses to cadmium stress in Arabidopsis thaliana

Abstract

Both historical and today’s industrial and agricultural activities contribute to the current worldwide pollution with toxic metals such as cadmium (Cd). Bioaccumulation of Cd poses a serious threat to the food chain, and therefore the entire ecosystem including human, animal and plant populations. Plants exposed to Cd suffer from disturbances in morphology (e.g. reduced crop yield) and physiology (e.g. photosynthesis). At the cellular level, Cd elicits an oxidative challenge, characterised by an elevated production of reactive oxygen species (ROS). Increased ROS levels have been associated with both damaging oxidising processes and protective signalling. The latter is suggested to activate the antioxidant system, which neutralises ROS and hence restores the redox equilibrium. This balance between ROS production and scavenging determines whether the oxidative challenge will lead to damage or signalling. Three major redox buffers in plant cells are involved in both signalling and antioxidant processes. These redox buffers consist of the reduced and oxidised forms of glutathione [i.e. glutathione (GSH) and glutathione disulfide (GSSG)], ascorbate [i.e. ascorbate (AsA) and dehydroascorbate (DHA)] and NADP (i.e. NADPH and NADP+ , respectively). Together with enzymes, these redox buffers constitute the AsA-GSH cycle, which is essential for the plant’s normal metabolism as well as its defence against oxidative stress. Under normal conditions, cells maintain highly reduced pools of these metabolites. Both GSH and AsA also have direct ROS scavenging capacity and serve as a substrate in other ROS detoxifying mechanisms. Besides these metabolites and their related enzymes, the superoxide -reducing superoxide dismutase (SOD) and hydrogen peroxide (H2O2)-neutralising catalase (CAT) are two major substrate-independent enzymes that complement the antioxidant system. In order to limit Cd-induced oxidative stress, plants additionally stimulate their chelation capacity. A primary Cd chelator that scavenges free damaging Cd ions, is phytochelatin (PC), a polymerised form of GSH. The thiol group on the cysteine residue of GSH causes this metabolite to function as signalling agent, antioxidant and Cd chelator. The current work focuses on GSH as well as AsA because they are both abundant, multifunctional and widely distributed metabolites in plant cells. The main goal of this research was to expand our knowledge concerning the role of GSH and AsA as chelator, antioxidant and/or redox buffer in Arabidopsis thaliana responses during moderate and severe, i.e. ranging from 1 to 10 µM, Cd exposure. To reveal spatiotemporal functions of GSH and AsA in early Cd detoxification, a kinetic screening was performed in wild-type plants. Cadmium chelation and antioxidative defences were investigated at different functional levels (gene expression, enzyme activity and metabolite content). The roots showed a fast but GSH-depriving PC production upon 2 h of Cd exposure. Despite SOD regulation, which already started after 2 h, alternative pathways including AsA and CAT were only activated after 24 h in order to complement the antioxidant function of GSH. Retention and detoxification of Cd in the roots contributed to a delayed response in the leaves. Together with high leaf GSH and AsA levels and possibly root-to-shoot signalling responses, the leaves had sufficient time to activate their defence mechanisms, preventing GSH deprivation. This experiment suggests a dual time shift, both within roots and between plant organs (Chapter 3). The use of multiple Cd concentrations (5 and 10 µM) and exposure times (2, 24, 48 and 72 h), provided the basis for more indepth research conducted in the following experiments. The use of A. thaliana as model organism, created the possibility to include mutants deficient in GSH and/or AsA. To gain more insight in the importance of these metabolites during the Cd-induced oxidative challenge, responses of wildtype plants were compared to mutants with 55 to 70% GSH reduction (cad2-1), 50 to 60% AsA reduction (vtc1-1) or both (cad2-1 vtc1-1) upon exposure to Cd during 24 (Chapter 4) and 72 h (Chapter 5). Both genotype- and Cd-specific responses concerning chelating (PC levels) and antioxidative mechanisms (gene expression, enzyme activity, metabolite redox state and content) were investigated. First, vtc1-1 mutants revealed an increased defence capacity under control conditions, including thiols (GSH and PC) and antioxidant transcripts [monodehydroascorbate reductase 3 (MDAR3) and glutaredoxin 480 (GRX480)]. In addition, these mutants did not demonstrate elevated oxidative stress at the level of GSH and AsA redox state or oxidative stress marker genes. Upon Cd exposure, enhanced PC synthesis appeared to efficiently compensate low AsA levels, contributing to a less Cd-sensitive genotype. Together, these findings suggest that AsA deficiency continuously primes vtc1-1 plants and therefore, prepares them to cope with additional stresses like exposure to Cd. Secondly, cad2-1 mutants were unable to compensate for their low metabolite levels. Under control conditions, these mutants revealed a more oxidised cellular GSH redox state and elevated expression of oxidative stress marker genes. The severe lack in GSH and PC levels resulted in a higher sensitivity to Cd in cad2-1 plants. The latter was indicated by Cd-induced activation of SOD, AsA and CAT pathways, which occurred to a greater extent in the cad2-1 mutants than wildtype plants. The current work suggests that GSH deficiency causes initial oxidative stress and the activation of multiple alternative pathways in order to cope with additional Cd stress. Based on data from the previous chapters, the role of GSH in SOD expression upon Cd exposure was investigated in a more in-depth experimental set-up (Chapter 6). Three mutants with GSH contents ranging from 15 to 45% of wildtype levels (cad2-1, pad2-1 and rax1-1) and wild-type plants were exposed to 5 µM Cd during 24 and 72 h and SOD expression was evaluated in all genotypes at transcriptional, posttranscriptional and enzyme activity level. In Cd-exposed wild-type plants, the activation of the squamosa promoter-binding protein-like 7 (SPL7) transcription factor resulted in up-regulation of FeSOD1 (FSD1) and microRNA398 (MIR398), which in its turn repressed CuZnSOD (CSD1 and CSD2) transcripts. Similar to wild-type plants, SPL7-dependent responses were activated in all Cd-exposed mutants, with the exception for both mature and primary CSD1/2 transcripts, which were significantly up-regulated in all GSH deficient mutants after 24 h. This fast but transient Cd-induced transcriptional activation of CSD transcripts after 24 h overruled the MIR398-regulated repression of CSD expression and resulted in increased SOD activity. This study suggests that the (subcellular) thiol redox state (concentration and/or reducedoxidised ratio) contributes to the transcription rate of CSD genes upon Cd exposure. Although the underlying mechanism remains unknown, a stillunidentified transcriptional activator was put forward to be responsible for this regulation. Finally, the importance of a PC-mediated response was investigated during longterm exposure to Cd (Chapter 7). Therefore, Cd and PC accumulation were followed in wild-type plants during three weeks of Cd exposure. Plant GSH and PC to Cd molar ratios (SH/Cd) initially increased, but after three days of Cd exposure, the ratios seemed to decrease over time. Also GSH deficient mutants (cad2-1, pad2-1 and rax1-1) were included to investigate the necessity of Cd- induced PC production for plant survival. Despite their increased Cd sensitivity, like the wild-type plants, all mutants were able to survive. These data suggest that after a fast PC response, including both Cd chelation and compartmentalisation, plants activate alternative mechanisms that complement the early Cd detoxification processes. In conclusion, all results support the essential role for GSH in early responses to Cd exposure. Both Cd chelation via PCs and ROS scavenging via SOD appeared to be a primary response in plants. Therefore, the lack of GSH and hence PCs, resulted in elevated activation of alternative antioxidant mechanisms including SOD, AsA and CAT. Moreover, this study suggests a regulatory role for GSH in SOD expression during Cd exposure, which might involve a still-unknown transcriptional activator. Long-term exposure to Cd however, indicated that the fast PC response was efficiently complemented by an alternative Cd detoxification mechanism.

Ter conclusie kunnen we stellen dat alle resultaten de essentiële rol voor GSH in de vroege respons van planten op Cd blootstelling ondersteunen. Zowel Cd chelatie via PCs als ROS neutralisatie via SOD bleken primaire reacties te zijn in planten. Hierdoor resulteerde het gebrek aan GSH, en bijgevolg PCs, in een verhoogde activatie van alternatieve antioxidatieve mechanismen inclusief SOD, AsA en CAT. Bovendien toont deze studie een regulerende rol voor GSH aan in de expressie van SOD tijdens blootstelling aan Cd, hetgeen de betrokkenheid van een nog steeds onbekende transcriptionele activator suggereert. Langdurige blootstelling aan Cd daarentegen, toonde aan dat de snelle reactie met PCs efficiënt werd aangevuld door een alternatief mechanisme van Cd detoxificatie.