Dissertation committee:
Ανδρέας Ρούσσης, Αναπληρωτής Καθηγητής Μοριακής Φυσιολογίας Φυτών, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Κοσμάς Χαραλαμπίδης, Αναπληρωτής Καθηγητής Μοριακής Βιολογίας Ανάπτυξης Φυτών, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Κώστας Θάνος, Ομότιμος Καθηγητής Φυσιολογίας Φυτών, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Σοφία Ριζοπούλου, Καθηγήτρια Φυσιολογίας Φυτών, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Βασίλειος Κουβέλης, Επίκουρος Καθηγητής Γενετικής και Βιοτεχνολογίας, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Εμμανουήλ Φλεμετάκης, Αναπληρωτής Καθηγητής Βιοχημείας Φυτών και Μικροοργανισμών, Τμήμα Βιοτεχνολογίας, Γεωπονικό Πανεπιστήμιο Αθηνών.
Κωνσταντίνος Βλαχονάσιος, Αναπληρωτής Καθηγητής Μοριακής Φυσιολογίας Φυτών, Τμήμα Βιολογίας, Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης.
Summary:
Selenium binding proteins (SBPs) are highly conserved and have representatives in all life kingdoms. In humans, many studies have demonstrated SBP1 importance in cancer development. In plants, SBP1 participates in biotic and abiotic stress responses. Previous studies have revealed its ability to interact with 14 proteins related to protein trafficking, membrane synthesis and redox control, indicating its participation in a novel protein network linked to oxidative stress. The aim of this thesis was to confirm the existence of the SBP1 network by studying the interacting proteins in the model plant Arabidopsis thaliana. Thus, two proteins related to protein trafficking (RD19c) and membrane synthesis (DALL3) were studied, while GRXS14, GRXS16 and SAH7 were additionally investigated for their ability to interact with SBP1. The phospholipase DALL3 and the protease RD19c were initially studied for tissue-specific expression, with the first one expressed in the root cylinder, hydothodes, guard cells, vasculature of cotyledons, first emerging leaves, sepals, siliques and anthers and in the stigma, while RD19c was expressed in guard cells, hydothodes, leaf trichomes, vasculature tissues of roots, lateral roots, first emerging leaves, cotyledons, sepals, anthers as well as in the silique base and in pollen. In these analyses it was shown that the transcipts of DALL3 and RD19c are expressed in the same tissues with those of SBP1, such as guard cells, stigma and root. Moreover, in subcellular analysis the phospholipase DALL3 was detected in the chloroplasts of the guard cells, in plastids of the root apex and the central root and in the lateral root primordia. Similarly, the protease localized in the root apex of the central and lateral roots and leaf trichomes. The relative expression levels were studied also in the allergen SAH7. The expression levels of these proteins were constitutive, with the highest expression in 10-day-old roots and flowers for DALL3 and minimum in 10-days-old seedlings and rosette leaves. The protease exhibited its highest expression levels in roots with no statistical significance difference and the lowest expression was in flowers and rosette leaves, while SAH7 was upregulated in cotyledons and shoots and downregulated in rosette leaves. Furthermore, the respective genes were studied for their induction in the presence of selenite, selenate and cadmium, and were highly expressed after selenite treatment, indicating their participation in the response mechanism related at least to this chemical compound. The initial investigation of protein-protein interactions was accomplished in yeast two-hybrid assay, which revealed the interaction of the four proteins with SBP1. In the same system, it was showed that the first 175 amino acids of SBP1 are crucial for its interaction with the DALL3 and RD19c, whereas the 105 with the GRXS14 and SAH7. Finally, the protease RD19c was detected in vesicles, the phospholipase DALL3 in speckle-like structures attached to chloroplasts and in root plastids, the gloutaredoxins GRXS14 and GRXS16 in chloroplasts, the allergen SAH7 in the endoplasmic reticulum and the SBP1 in cytoplasm and nucleus. Despite their different subcellular localization we revealed that these interactions can take place in planta. SBP1 interacts with the DALL3 in speckle-like structures attached to the chloroplasts and in root plastids, with the RD19c in cytoplasm, GRXS14 in the cytoplasm and nucleus, GRXS16 only in cytoplasm and lastly SAH7 in the endoplasmic reticulum. SBP1 is known for its participation in plant detoxification procedures against oxidative stress and our study confirms the existence of a novel protein network, where SBP1 exhibits the main role and is related to plant responses against oxidative stress.
Keywords:
Selenium binding proteins, Phosphlipases, Proteases, Allergens, Glοutaredoxins, Interactions, Stress, Protein network
