Biosíntesis de tioles y fitoquelatinas en leguminosas modelo

Abstract

he general objective of this Thesis is to study in detail the biosynthetic pa thway of thiols and (homo)phytochelatins in legumes. To this purpose, we will preferentially use Lotus japonicus as a model legume. The pathway for the biosynthesis of thiol compounds and phytochelatins in plants starts with the synthesis of cysteine (Fig. 1, in grey). The synthesis of this essential amino acid is catalyzed by cysteine synthase, a complex formed by two enzymes: serine-acetyltransferase and o-acetylserine(thiol)lyase. Cysteine is then used as a precursor of the thiol tripeptide glutathione (γGlu-Cys-Gly), which is formed by the sequential action of two ATP-dependent enzymes. First, γ−glutamylcysteine synthetase produces the thiol dipeptide γ−glutamylcysteine, and second, glutathione synthetase catalyzes the formation of glutathione by adding a glycine residue to the C-terminus of γ−glutamylcysteine. In plants exposed to heavy metals, glutathione serves as a precursor of phytochelatins, which are cysteine-rich polypeptides that bind certain metals with high affinity. The phytochelatin-metal complexes are transported into the vacuoles, thus avoiding toxic effects of metals on metabolism.

Leguminous plants have the peculiarity that they are able to synthesize homoglutathione (γGlu-Cys-βAla) in addition to, or instead of, glutathione (Fig. 1, in yellow). However, it is unclear whether homoglutathione is formed by a glutathione synthetase with broad substrate specificity or by a specific homoglutathione synthetase. Likewise, legumes synthesize homophytochelatins, structural phytochelatin homologs, from homoglutathione. It is unknown whether these polypeptides are synthesized by typical phytochelatin synthases or by a specific homophytochelatin synthase.

We have therefore set up this work with the following four specific objectives:

(1) To elucidate whether homoglutathione in legumes is synthesized by glutathione synthetase or by a specific homoglutathione synthetase, and, in the second case, to characterize the corresponding genes and proteins.

(2) To identify, map, and characterize the thiol synthethase genes of L. japonicus. This objective will include the genes encoding γ−glutamylcysteine synthetase, glutathione synthetase, and, if applicable, homoglutathione synthetase.

(3) To study the regulatory mechanisms of the pathway for thiol and (homo)phytochelatin biosynthesis in L. japonicus plants exposed to heavy metals. This experimental treatment will be used to induce mobilization of thiol compounds, in particular cysteine and (homo)glutathione, for (homo)phytochelatin synthesis.

(4) To characterize, at the molecular and biochemical levels, phytochelatin synthases of L. japonicus.