Flavonoid oxidation in Arabidopsis thaliana seeds. Functional analysis of the TRANSPARENT TESTA 10 gene encoding a polyphenoloxidase of the laccase type.

Abstract : Arabidopsis seeds accumulate flavonoids (proanthocyanidins and flavonols) during their development. A previous study has shown that a laccase (AtLAC15) encoded by the TRANSPARENT TESTA 10 (TT10 ) gene could trigger flavonoid oxidation in the seed coat. If both proanthocyanidins (PAs) and flavonols appear to be TT10 protein substrates, only PA oxidation leads to brown pigments responsible for the mature seed coat color. An important consequence of TT10 activity on seed flavonoid metabolism is an increased ratio of insoluble to soluble PAs. The physiological functions of TT10 are still unknown, however defense against biotic and abiotic stresses, either constitutive or induced, may be predicted on the basis of present knowledge on polyphenoloxidases. The purpose of this thesis was to perform a functional characterization of the TT10 gene. A part of the work was devoted to the analysis of the regulatory mechanisms controlling the developmental pattern of TT10 gene expression in seeds and vegetative plant parts. The functional 5’-dissection of a 2.0-kb promoter realized with the uidA reporter gene encoding β-glucuronidase (GUS) was performed to identify regions responsible for activation in seed and other plant organs. TT10 promoter happens to be activated exclusively in seed coat and siliques. Directed mutagenesis was undertaken to precise the regulatory role of in silico-detected cis-acting regulatory elements (CAREs) located in a 194-bp region necessary for expression in seed coat. TT10 gene expression assessed in different tissues at various stages of development using qRT-PCR matched promoter activity pattern. Natural variation for TT10 expression among Arabidopsis accessions was also detected, with the levels of TT10 mRNA in Cvi, Ler and Sha being strongly reduced compared to the ones in Ws, Col and Bay. The impact of this molecular polymorphism on seed flavonoid composition, as analyzed on mature seeds with LC-MS, is discussed. In silico analysis of the TT10 promoter revealed the presence of putative CAREs potentially involved in signaling and response to biotic and abiotic stresses. However histochemical analysis of GUS activity in transgenic Arabidopsis plantlets expressing pT T102.0−kb : GUS failed to detect any ectopic activity when submitted to a variety of stresses. This result suggests that transcriptional response to environmental stimuli is highly constrained by developmental parameters. TT10 appeared to be the only member of the laccase gene family to be strongly expressed in seeds. TT10 function may have evolved towards flavonoid oxidation by co-localization with these substrates, which is ensured by tissue-specific gene expression.
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Przemyslaw Bidzinski. Flavonoid oxidation in Arabidopsis thaliana seeds. Functional analysis of the TRANSPARENT TESTA 10 gene encoding a polyphenoloxidase of the laccase type.. Molecular biology. Universite Paris XI Orsay, 2009. English. ⟨tel-02177497⟩

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