The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B" (a regulatory) subunits. The c3c4 (C subunit) and fass (B") subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B" subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B" subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.

To evaluate the effects of the cyanobacterial toxin microcystin-LR (MCY-LR, a protein phosphatase inhibitor) and diquat (DQ, an oxidative stress inducer) on the organization of tonoplast, the effect of MCY-LR on plastid stromule formation and on mitochondria was investigated in wild-type Arabidopsis. Tonoplast was also studied in PP2A catalytic (c3c4) and regulatory subunit mutants (fass-5 and fass-15). These novel studies were performed by CLSM microscopy. MCY-LR is produced during cyanobacterial blooms. The organization of tonoplast of PP2A mutants of Arabidopsis is much more sensitive to MCY-LR and DQ treatments than that of wild type. In c3c4, fass-5 and fass-15, control and treated plants showed increased vacuole fragmentation that was the strongest when the fass-5 mutant was treated with MCY-LR. It is assumed that both PP2A/C and B” subunits play an important role in normal formation and function of the tonoplast. In wild-type plants, MCY-LR affects mitochondria. Under the influence of MCY-LR, small, round-shaped mitochondria appeared, while long/fused mitochondria were typical in control plants. Presumably, MCY-LR either inhibits the fusion of mitochondria or induces fission. Consequently, PP2A also plays an important role in the fusion of mitochondria. MCY-LR also increased the frequency of stromules appearing on chloroplasts after 1 h treatments. Along the stromules, signals can be transported between plastids and endoplasmic reticulum. It is probable that they promote a faster response to stress.

Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses.

The serine-threonine protein phosphatase PP2A holoenzymes regulate many eukaryotic cellular processes. They consist of catalytic (C), regulatory (B) and scaffolding (A) subunits. C3-C4 and their interacting B" (Fass) subunits of PP2A were in the focus of this study. Our tools were Arabidopsis wild-type plants and phosphatase mutants-fass (B"), c3c4 and microcystin-LR (MCY-LR), a specific inhibitor of PP2A/PP1. We have studied their role in the regulation of protein phosphatase activities and expression. In relation to this, we have focused on their involvement in mitotic activity and histone H3 phosphorylation. We show here for the first time that besides C3-C4, B"/Fass subunit has a crucial role in the maintenance of normal protein phosphatase activities and levels in Arabidopsis. fass mutants show increased activities of PP2A and PP1 (a phosphatase related to PP2A) and increased expression of PP2A/C. Both overall PP2A and PP1 activities are inhibited in c3c4. Fass and C3-C4 are involved in the regulation of mitotic activity. C3-C4 have less direct influence on the onset of mitosis, but Fass is involved in the regulation of metaphase-anaphase transition. In wild-type plants, MCY-LR inhibited PP2A/C activities, related to alterations of mitotic events. Distinct effects of MCY-LR in mutants also reveal the role of Fass/C3-C4 in the biochemical functionality of PP2A. Fass and C3-C4 regulate the phosphorylation of histone H3, but this effect is not related to the regulation of mitotic onset in Arabidopsis. This study gives new insights into the regulatory roles of Fass and C3-C4 subunits during mitosis in plants.