Apoplastic glutathione degradation by gamma-glutamyl transferase isoforms GGT1 and GGT2 is important for generative and vegetative development in Arabidopsis thaliana
Gamma-glutamyl transferase (GGT) is the unique enzyme able to degrade glutathione (GSH) in extra cytosolic spaces. In plant cell GGT1 and GGT2 isoforms are located in the apoplast, bound to the cell wall and to the plasma membrane respectively. GGT1 is expressed in whole plants mainly in leaves and in the vascular system, whereas more specific expression concerns GGT2 that is found in seed, trichome, pollen and weakly in root. However their role in plant physiology is still waiting to be defined. Glutathione, the substrate of GGT, is one of the most multifunctional molecule in biology, being the most important redox buffer and in plants one of the major source of reduced sulfur. Glutathione is a constituent of the phloem sap, but the mechanisms of its phloem loading and unloading are largely unknown. Obtaining and characterizing the ggt1/ggt2 RNAi double mutant allows to add more clues than the relative single mutants and to shutdown possible compensatory expression between the two isoforms. In this work the selection and characterization of previously obtained ggt1/ggt2 RNAi lines has been performed. The silencing level of both GGT1 and GGT2 transcript was verified by qRT-PCR and the total GGT activity analyzed. Furthermore phenotypic characterization was carried out; firstly, the GGT contribution in cysteine delivery to the seed and in seed development and composition was investigated. The ggt1/ggt2 RNAi plants showed lower seed yield due to reduced silique number and silique length. However thiol content and 2S albumin storage proteins did not change in mutant seeds. It can be concluded that GGT silencing results in metabolic readjustments leading to allocation of resources to less, but fully viable seeds. The fact that GGT uses GSH as a substrate poses the question whether altered resource allocation results from impairment of cysteine delivery to sink tissues and cells, or from redox imbalances which may result in altered glutathione metabolism. The latter might correlate with the observed proline accumulation, which suggests the occurrence of oxidative stress affecting also the germination rate, which resulted delayed. The vegetative growth was also slightly slowed down with a reduced growth rate of rosette diameter and root length. Trichomes, the leaf hair characterized by high level of GSH, were also less in number than wild-type. Moreover the GSH depletion by roots in ggt mutants suggests cooperation between the two isoforms. In conclusion the simultaneous GGT1 and GGT2 silencing induces the decrease of number of organs with high GSH demand (seed and trichome), oxidative stress in seeds and slightly affects vegetative growth. Further information about GGT will be provided by the double mutants obtained in this thesis work through the crossing between ggt mutants with sir1-1, that has reduced sulfur flux, and with ggct2;1, that lacks cytosolic GSH degradation
