The one and only enzyme able to breakdown glutathione (GSH) in extracellular spaces is gamma-glutamyl transferase (GGT). GGT1 and GGT2 isoforms are found in the apoplast of plant cells, where they are linked to the cell wall and the plasma membrane. GGT1 is expressed throughout the plant, primarily in the leaves and vascular system, whereas GGT2 is found in seed, trichome, pollen, and, to a lesser extent, the root. Their involvement in plant physiology, however, is still unknown. Glutathione, the GGT substrate, is one of the most versatile molecules in biology, serving as the most essential redox buffer and a key source of reduced sulfur in plants. Glutathione is a component of phloem sap, but the mechanisms governing its phloem loading and unloading are poorly understood. Obtaining and characterizing the ggt1/ggt2 RNAi double mutant adds more clues than the related single mutants and eliminates the possibility of compensatory expression between the two isoforms. This study focused on the selection and characterization of previously acquired ggt1/ggt2 RNAi lines. qRT-PCR was used to confirm the silencing level of both GGT1 and GGT2 transcripts, and total GGT activity was measured. Additionally, phenotypic characterization was performed by measuring the dry weight of seed and leave morphology. In this study most of work was performed on GGT1 Mutant. The iTRAQ-based quantitative proteomic analysis of the seeds of GGT1 mutants revealed that the abundance of 41 DAPs increased with more than 2-fold increase in 7 DAPs, while 34 DAPs had decreased abundance. Functional categorization of the DAPs revealed that the most altered biological processes were primary and secondary metabolism, response to stress, redox homeostasis, protein transport, folding and proteolysis, and seed storage. The abundance of proteins related to energy production and conversion, transport and metabolism of amino acids, lipids and inorganic ions was most altered.
The one and only enzyme able to breakdown glutathione (GSH) in extracellular spaces is gamma-glutamyl transferase (GGT). GGT1 and GGT2 isoforms are found in the apoplast of plant cells, where they are linked to the cell wall and the plasma membrane. GGT1 is expressed throughout the plant, primarily in the leaves and vascular system, whereas GGT2 is found in seed, trichome, pollen, and, to a lesser extent, the root. Their involvement in plant physiology, however, is still unknown. Glutathione, the GGT substrate, is one of the most versatile molecules in biology, serving as the most essential redox buffer and a key source of reduced sulfur in plants. Glutathione is a component of phloem sap, but the mechanisms governing its phloem loading and unloading are poorly understood. Obtaining and characterizing the ggt1/ggt2 RNAi double mutant adds more clues than the related single mutants and eliminates the possibility of compensatory expression between the two isoforms. This study focused on the selection and characterization of previously acquired ggt1/ggt2 RNAi lines. qRT-PCR was used to confirm the silencing level of both GGT1 and GGT2 transcripts, and total GGT activity was measured. Additionally, phenotypic characterization was performed by measuring the dry weight of seed and leave morphology. In this study most of work was performed on GGT1 Mutant. The iTRAQ-based quantitative proteomic analysis of the seeds of GGT1 mutants revealed that the abundance of 41 DAPs increased with more than 2-fold increase in 7 DAPs, while 34 DAPs had decreased abundance. Functional categorization of the DAPs revealed that the most altered biological processes were primary and secondary metabolism, response to stress, redox homeostasis, protein transport, folding and proteolysis, and seed storage. The abundance of proteins related to energy production and conversion, transport and metabolism of amino acids, lipids and inorganic ions was most altered.
Proteomic and biochemical analyses of Arabidopsis thaliana mutants in glutathione metabolism
MAQSOOD, MUHAMMAD IMRAN
2022/2023
Abstract
The one and only enzyme able to breakdown glutathione (GSH) in extracellular spaces is gamma-glutamyl transferase (GGT). GGT1 and GGT2 isoforms are found in the apoplast of plant cells, where they are linked to the cell wall and the plasma membrane. GGT1 is expressed throughout the plant, primarily in the leaves and vascular system, whereas GGT2 is found in seed, trichome, pollen, and, to a lesser extent, the root. Their involvement in plant physiology, however, is still unknown. Glutathione, the GGT substrate, is one of the most versatile molecules in biology, serving as the most essential redox buffer and a key source of reduced sulfur in plants. Glutathione is a component of phloem sap, but the mechanisms governing its phloem loading and unloading are poorly understood. Obtaining and characterizing the ggt1/ggt2 RNAi double mutant adds more clues than the related single mutants and eliminates the possibility of compensatory expression between the two isoforms. This study focused on the selection and characterization of previously acquired ggt1/ggt2 RNAi lines. qRT-PCR was used to confirm the silencing level of both GGT1 and GGT2 transcripts, and total GGT activity was measured. Additionally, phenotypic characterization was performed by measuring the dry weight of seed and leave morphology. In this study most of work was performed on GGT1 Mutant. The iTRAQ-based quantitative proteomic analysis of the seeds of GGT1 mutants revealed that the abundance of 41 DAPs increased with more than 2-fold increase in 7 DAPs, while 34 DAPs had decreased abundance. Functional categorization of the DAPs revealed that the most altered biological processes were primary and secondary metabolism, response to stress, redox homeostasis, protein transport, folding and proteolysis, and seed storage. The abundance of proteins related to energy production and conversion, transport and metabolism of amino acids, lipids and inorganic ions was most altered.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/49904