Are Methionine Sulfoxide-Containing Proteins Related to Seed Longevity? A Case Study of Arabidopsis thaliana Dry Mature Seeds Using Cyanogen Bromide Attack and Two-Dimensional-Diagonal Electrophoresis

Abstract

In recent years, several reports pointed out the role of protein oxidation in seed longevity, notably regarding the oxidation of methionine (Met) residues to methionine sulfoxide (MetO) in proteins. To further consider this question, we present a handy proteomic method based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage, which we refer to as 2Dd-CNBr. CNBr treatment of proteins causes the non-enzymatic hydrolysis of peptide bonds on the carboxyl side of reduced Met residues. However, Met oxidation causes a lack of cleavage, thus modifying the electrophoretic mobility of CNBr-induced peptides. This approach was first validated using bovine serum albumin as a model protein, which confirmed the possibility of distinguishing between oxidized and non-oxidized forms of Met-containing peptides in gels. Then, the 2Dd-CNBr method was applied to the Arabidopsis thaliana seed protein extract in a control (non-oxidized) condition and in an oxidized one (as obtained following hypochlorous acid treatment). Twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins. In the three-dimensional environment of the oxidized Met, we detected amino acid residues that could be converted by oxidation (carbonylation) or by phosphorylation, suggesting a possible interplay between Met oxidation and the other protein modifications. The identification of the proteins oxidatively modified in Met residues revealed the finding that MetO-containing proteins are related to seed longevity. Based on these results, we suggest that the method presently described also has the potential for wider applications.

The association of protein-bound methionine sulfoxide with proteomic basis for aging in beech seeds

“…An increased abundance of MetO peptides was confirmed for the dehydrin protein, while three LEA proteins (Lea1, D-34, D-29) were reported to display declining MetO levels under long-term storage. LEAs were reported to contain MetO already in mature dry (non-stored) seeds of Arabidopsis [ 65 ]. Predominantly, exposed Met residues are oxidized in properly folded and functional proteins [ 66 ], as well as intrinsically disordered proteins [ 27 ], including LEAs [ 67 ].…”

“…Predominantly, exposed Met residues are oxidized in properly folded and functional proteins [ 66 ], as well as intrinsically disordered proteins [ 27 ], including LEAs [ 67 ]. All oxidized Met residues identified in proteins, including several LEAs, extracted from dry Arabidopsis seeds not subjected to stress conditions, were surface exposed [ 65 ]. Disordered regions of proteins and intrinsically disordered proteins are highly prone to oxidation [ 27 ].…”

Recent progress in molecular genetics and omics-driven research in seed biology

“…Then, a more severe oxidative attack of Met results in the irreversible formation of a sulfone derivative (Met sulfone). In a recent work carried out on mature dry seeds of Arabidopsis, twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins [209]. In the three-dimensional environment of the oxidized Met, amino acid residues were detected that could be converted by oxidation (carbonylation) or by phosphorylation suggesting a possible interplay between Met oxidation and the other PTMs.…”

“…Studies carried out since 2010 illustrate that ROS and RNS are performing oxidative and nitrosative signalling to induce seed germination within an oxidative window level [201,[208][209][210]. ROS/RNSmediated post-translational modifications (PTMs) such as carbonylation, oxidation of the sulfur amino acids, S-nitrosylation and nitration can promote seed germination.…”

“…Une attaque oxydative plus sévère de Met entraîne la formation irréversible d'un dérivé sulfonique (Met sulfone). Dans un travail récent réalisé sur des graines sèches matures d'Arabidopsis, vingt-quatre résidus Met oxydés ont été identifiés dans 19 protéines par spectrométrie de masse [209]. Des résidus d'acides aminés convertis par oxydation (carbonylation) ou par phosphorylation ont été détectés dans l'environnement tridimensionnel des Met oxydés, ce qui suggère une interaction possible entre l'oxydation de résidus Met et d'autres PTMs.…”