Overexpression of the Arabidopsis glutathione peroxidase-like 5 gene (AtGPXL5) resulted in altered redox status, plant development and salt tolerance Ph.D. Dissertation Riyazuddin Riyazuddin Supervisor: Dr. Jolán Csiszár Associate professor Doctoral School of Biology Department of Plant Biology University of Szeged, Faculty of Science and Informatics Közép fasor 52., H-6726 Szeged, Hungary 2020 Szeged Table of Contents 1. List of abbreviations ......................................................................................................... 3 2. Introduction ....................................................................................................................... 4 2.1 The main environmental stresses and the effect of excess salt on plants ............................................... 4 2.2 Reactive oxygen species (ROS), oxidative stress, and their challenges ................................................ 6 •− Superoxide radical anion (O2 ) ......................................................................................................... 7 Hydrogen peroxide (H2O2) ................................................................................................................ 8 Hydroxyl radical (•OH) ..................................................................................................................... 8 2.3 Lipid peroxides (LP) .............................................................................................................................. 8 2.4 Non-enzymatic antioxidant defense system......................................................................................... 10 Ascorbic acid (AsA) ........................................................................................................................ 10 Glutathione (GSH) .......................................................................................................................... 11 Glutathione half-cell reduction potential ......................................................................................... 11 2.5 Enzymatic antioxidant defense system ................................................................................................ 12 Superoxide dismutase (SOD; EC 1.15.1.1.) .................................................................................... 12 Catalase (CAT; EC 1.11.1.6) ........................................................................................................... 13 Enzymes of the ascorbate-glutathione (AsA-GSH) cycle ............................................................... 13 Guaiacol peroxidase (POX; EC: 1.11.1.7) ...................................................................................... 14 Glutathione transferases (GSTs; EC 2.5.1.18) ................................................................................ 15 Thiol-based peroxidases .................................................................................................................. 15 2.6 Glutathione peroxidases (EC 1.11.1.9, EC 1.11.1.12 and EC 1.11.1.15) ............................................ 16 3. Aims .................................................................................................................................. 25 4. Materials and methods ................................................................................................... 26 4.1 Plant material ....................................................................................................................................... 26 Growth conditions and stress treatments ......................................................................................... 26 4.2 Cloning of the AtGPXL5 gene and the used vector constructions ....................................................... 27 4.3 RNA extraction, expression analyses with quantitative real-time PCR (qRT-PCR) ........................... 29 4.4 Detection of ROS levels and vitality ................................................................................................... 30 4.5 Investigation of the growth and development of seedlings .................................................................. 30 4.6 Analysis of some stress markers .......................................................................................................... 33 4.7 Evaluation of the AsA and GSH contents ........................................................................................... 34 4.8 Calculation of the glutathione half-cell reduction potential ................................................................. 35 4.9 Antioxidant enzyme activity measurements ........................................................................................ 35 4.10 Statistical analysis................................................................................................................................ 37 5. Results .............................................................................................................................. 38 5.1 Investigation of the role of AtGPXL5 in ROS homeostasis using the mutant seedlings ..................... 38 Effect of the mutation of AtGPXL5 on the ROS levels and vitality of seedlings ............................ 38 5.2 Overexpression of AtGPXL5 gene ....................................................................................................... 42 The germination rate of the AtGPXL5-overexpressing seeds was less inhibited under salt stress .. 43 1 The growth of roots of AtGPXL5-overexpressing lines under salt stress condition was more retained 44 Developmental traits of shoots under salt stress .............................................................................. 45 Image analysis of shoots indicate less affected chlorophyll and anthocyanin contents in overexpressed plants under salt stress .......................................................................................................... 47 5.3 Overexpression of AtGPXL5 influences some stress-related parameters in adult plants ..................... 48 Expression level of AtGPXL5 in the root and shoot of Arabidopsis plants with or without salt treatment ....................................................................................................................................................... 48 Thioredoxin peroxidase and glutathione peroxidase activities of the overexpressed Arabidopsis plants are almost like in the wild type plants ................................................................................................ 49 Hydrogen peroxide content and lipid peroxide level of plants ........................................................ 50 H2O2-related enzyme activities in the wild type and transgenic Arabidopsis plants ....................... 51 GSH-related antioxidant enzymatic activities of plants .................................................................. 53 5.4 The ascorbate and glutathione levels and their reduced status ............................................................ 54 6. Discussion ......................................................................................................................... 56 6.1 Role of plant GPXLs in stress tolerance .............................................................................................. 56 6.2 AtGPXL5 isoenzyme has a key role in the regulation of the ROS homeostasis and in the maintenance of the cell’s vitality in the Arabidopsis thaliana seedlings ............................................................................... 57 6.3 The expression level of AtGPXL5 gene can influence the main antioxidant mechanisms in the short- term salt stress condition .................................................................................................................................. 58 6.4 AtGPXL5 affect the redox homeostasis by the enhancement of reduced glutathione and thus the reduction potential becomes more negative than in the wild type .................................................................... 59 6.5 AtGPXL5 has a role in the seed germination, growth and development of Arabidopsis seedlings under control and in the presence of 100 mM NaCl ................................................................................................... 60 7. Summary .......................................................................................................................... 64 8. Összefoglalás .................................................................................................................... 66 9. Acknowledgements ......................................................................................................... 68 10. References ........................................................................................................................ 70 11. Publication list ............................................................................................................... 101 12. Supplementary data ...................................................................................................... 106 2 1. List of abbreviations ABA: abscisic acid LP: lipid peroxides APX: ascorbate peroxidase MDA: malondialdehyde AsA: reduced ascorbate MDHA: monodehydroascorbate CaMV35S: cauliflower mosaic virus 35S MDHAR: monodehydroascorbate reductase promoter MES/KCl: 2-(N-Morpholino) ethanesulfonic CAT: catalase acid/ potassium chloride buffer CDNB: 1-chloro-2,4-dinitrobenzene MS: Murashige-Skoog medium CHP: cumene hydroperoxide NTR: NADPH-dependent