Two class III peroxidases from Arabidopsis, AtPrx33 and Atprx34, have been studied in this paper. Their encoding genes are mainly expressed in roots; AtPrx33 transcripts were also found in leaves and stems. Light activates the expression of both genes in seedlings. Transformed seedlings producing AtPrx33-GFP or AtPrx34-GFP fusion proteins under the control of the CaMV 35S promoter exhibit fluorescence in the cell walls of roots, showing that the two peroxidases are localized in the apoplast, which is in line with their affinity for the Ca(2+)-pectate structure. The role they can play in cell wall was investigated using (1) insertion mutants that have suppressed or reduced expression of AtPrx33 or AtPrx34 genes, respectively, (2) a double mutant with no AtPrx33 and a reduced level of Atprx34 transcripts, (3) a mutant overexpressing AtPrx34 under the control of the CaMV 35S promoter. The major phenotypic consequences of these genetic manipulations were observed on the variation of the length of seedling roots. Seedlings lacking AtPrx33 transcripts have shorter roots than the wild-type controls and roots are still shorter in the double mutant. Seedlings overexpressing AtPrx34 exhibit significantly longer roots. These modifications of root length are accompanied by corresponding changes of cell length. The results suggest that AtPrx33 and Atprx34, two highly homologous Arabidopsis peroxidases, are involved in the reactions that promote cell elongation and that this occurs most likely within cell walls.
Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H2O2 modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H2O2 in different light qualities. H2O2 either promoted germination or repressed germination depending on the light wavelengths, showing that H2O2 acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H2O2-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.
An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.
An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca 2 ؉ -induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca 2 ؉ -pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca 2 ؉ -pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.
The high number of peroxidase genes explains the description of numerous physiological functions and the fact that the in planta function of a single isoform has never been characterized yet. We analyzed in transgenic Arabidopsis thaliana the localization of a zucchini isoperoxidase (APRX), previously puriWed thanks to its pectin binding ability. We conWrmed that the protein is localized near the cell wall, mainly produced in the elongation area of the hypocotyls and respond to exogenous auxin. In addition, the ectopic overexpression of APRX induced changes in growth pattern and a signiWcant reduction of endogenous indole-3-acetic acid (IAA) level. In agreement with these observations APRX showed an elevated in vitro auxin oxidase activity. We propose that APRX participates in the negative feedback regulation of auxin level and consequently terminates the hypocotyl elongation process.
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