Origin, evolution and targeting of THI1 protein in plants.

Author(s)

Juliana Dantas de Almeida

• Document type: Doctoral Thesis
• Press: Piracicaba. , ilustrações, tabelas.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2004-04-13

Examining board members

Marcio de Castro Silva Filho; Maria Helena de Souza Goldman; Ivan de Godoy Maia; Francisco Gorgonio da Nobrega; Marie Anne van Sluys

• Advisor: Marcio de Castro Silva Filho
• Field of knowledge: Biological Sciences - Biochemistry
• Indexed in: Banco de Dados Bibliográficos da USP-DEDALUS; Biblioteca Digital de Teses e Dissertações - USP
• Location: Universidade de São Paulo. Biblioteca Central da Escola Superior de Agricultura Luiz de Queiroz; ESALQ-BC t575.1; A447o; 83154

Abstract

THI1 is probably a bifunctional protein, since it is involved in thiamin biosynthesis and organelar genome stability mainly the mitochondrial. Interestingly, the thiamin biosynthesis occurs at different compartments in plants (chloroplasts) and yeasts (mitochondria). Functional complementation assays showed that Arabidopsis thaliana thi1 gene is able to complement a yeast mutant strain for the hortolog gene. The Arabidopsis thaliana THI1 is encoded by a single copy gene. A detailed analysis of the THI1 N-terminal region, that is responsible for its targeting in cells, reveled the presence of two in tanden directing sequences. At N-terminal region there is a chloroplastic transit peptide followed by a region able to form an anfifilic α-helix frequently present in mitochondrial presequences. Aiming to evaluate if the THI1 final localization could present a temporal or spacial regulation, transgenic plants expressing THI1 fused to GFP ("green fluorescent protein") where obtained. Confocal microscopy analysis of these transgenic plants showed that THI1 is mainly found in chloroplasts and barely found in mitochondrias. Different from what happens in Arabidopsis thaliana, in sugar cane where founded at least three thi1 isoforms/paralogs. The amino acids sequence alignment of these isoforms with the thi1 one, reveled high similarity including the targeting sequence. To evaluate the directing standard of these sugarcane isoforms, gene constructions made by the complete targeting sequence or the chloroplastic transit peptide or the mitochondrial presequence, fused to GFP under the guidance of 35S promotor, where obtained. A transient expression of these gene constructios in epidermal onion cells prove that in the case of the constructions containing either the complete targeting sequence or the chloroplastic transit peptide the directing occurred only to chloroplasts. On the other hand, the constructions containing the mitochondrial pre sequence, GFP were kept defused in the citoplasm. Besides the directing aspect, THI1 were evaluated under the filogenetic point of view. Filogenetic analysis showed that thi1 is rarely found in bacteria but is widely distributed in Archaea. The genetic distances pointed out that probably eucaryotes THI1 came from Archaea. This gene in a few bacterias probably were inherited by lateral transference. (AU)