Dataset Information

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

ABSTRACT: Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from ¹H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

SUBMITTER: Caccamo A

PROVIDER: S-EPMC10669542 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Caccamo Anna A Vega de Luna Félix F Wahni Khadija K Volkov Alexander N AN Przybyla-Toscano Jonathan J Amelii Antonello A Kriznik Alexandre A Rouhier Nicolas N Messens Joris J Remacle Claire C

Antioxidants (Basel, Switzerland) 20231031 11

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from <i>Chlamydomonas reinhardtii</i> referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arg ...[more]

PMID: 38001799

Similar Datasets

Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Experiment Overall Design: Arabidopsis thaliana rosette leaves were harvested after 2 h of reillumination following a 8h dark period for RNA extraction and hybridization on Affymetrix ATH1 microarrays. The entire experiment was performed six times, providing independent biological replicates. For each of the six experiments, all four lines, wild-type, thylakoidal ascorbate peroxidase overexpressor (over tAPX, line 14/2), flu mutant and flu plants overexpressing thylakoidal ascorbate peroxidase were grown for 3 weeks under continuous light at 90 mmol. m-2 . s-1, transferred to the dark for 8 h, and reilluminated for 120 min before the rosette leaves of at least 10 plants per line were harvested. Total RNAs from three separate biological experiments were pooled (= 1 biological rep.) for the preparation of cDNA and the subsequent synthesis of biotin-labeled complementary RNA as recommended by Affymetrix.

Project description:Nitric oxide (NO) is a free radical which modulates protein function and gene expression throughout all stages of plant development. Fruit ripening involves a complex scenario where drastic phenotypical and metabolic changes take place. Pepper fruits are one of the most consumed horticultural products worldwide which, at ripening, undergo crucial phenotypical and biochemical events, with NO and antioxidants being implicated. Based on previous transcriptomic (RNA-Seq), proteomics (iTRAQ), and enzymatic data, this study aimed to identify the ascorbate peroxidase (APX) gene and protein profiles in sweet peppers and to evaluate their potential modulation by NO during fruit ripening. The data show the existence of six CaAPX genes (CaAPX1-CaAPX6) that encode corresponding APX isozymes distributed in cytosol, plastids, mitochondria, and peroxisomes. The time course expression analysis of these genes showed heterogeneous expression patterns throughout the different ripening stages, and also as a consequence of treatment with NO gas. Additionally, six APX isozymes activities (APX I-APX VI) were identified by non-denaturing PAGE, and they were also differentially modulated during maturation and NO treatment. In vitro analyses of fruit samples in the presence of NO donors, peroxynitrite, and glutathione, showed that CaAPX activity was inhibited, thus suggesting that different posttranslational modifications (PTMs), including S-nitrosation, Tyr-nitration, and glutathionylation, respectively, may occur in APX isozymes. In silico analysis of the protein tertiary structure showed that residues Cys32 and Tyr235 were conserved in the six CaAPXs, and are thus likely potential targets for S-nitrosation and nitration, respectively. These data highlight the complex mechanisms of the regulation of APX isozymes during the ripening process of sweet pepper fruits and how NO can exert fine control. This information could be useful for postharvest technology; NO regulates H2O2 levels through the different APX isozymes and, consequently, could modulate the shelf life and nutritional quality of pepper fruits.

Dataset Information

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Publications

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets