Protein phosphatase PP2A is an enzyme complex consisting of C (catalytic), A (scaffold) and B (regulatory) subunits. B subunits are a large family of proteins that regulate activity, substrate specificity and subcellular localization of the holoenzyme. Knowledge on the molecular functions of PP2A in plants is less than for protein kinases, but it is rapidly increasing. B subunits are responsible for the large diversity of PP2A functioning. This paper intends to give a survey on their multiple regulatory mechanisms. Firstly, we give a short description on our current knowledge in terms of “B”-mediated regulation of metabolic pathways. Next, we present their subcellular localizations, which extend from the nucleus to the cytosol and membrane compartments. The next sections show how B subunits regulate cellular processes from mitotic division to signal transduction pathways, including hormone signaling, and then the emerging evidence for their regulatory (mostly modulatory) roles in both abiotic and biotic stress responses in plants. Knowledge on these issues should be increased in the near future, since it contributes to a better understanding of how plant cells work, it may have agricultural applications, and it may have new insights into how vascular plants including crops face diverse environmental challenges.

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.

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.

Summary: Posttranslational modification of histones during the cell cycle is a major process controlling many aspects of cell division. Among the variety of histone modifications, mitotic phosphorylation of histone H3 at serine 10 (H3S10ph) plays a crucial role, particularly in proper chromosome segregation. Here we aimed at precisely quantifying this phosphorylation dynamics during mitosis in plant cells in order to reveal molecular pathways involved in this process. We describe an analysis pipeline based on 3D image analysis that allows to semiautomatically quantify H3S10ph in mitotic Arabidopsis root cells. We also developed a new method for the compensation of signal attenuation in Z, based on measurement of objects of interest themselves. We show that this new attenuation correction method allows significant gains in accuracy and statistical power. Using this pipeline, we were able to reveal small H3S10ph differences between cells treated with hesperadin, an inhibitor of an H3S10ph kinase, or between Arabidopsis mutants affected in PP2A phosphatase activity. This tool opens new avenues to explore such regulatory pathways in plants, using the wealth of genetic materials available in Arabidopsis. It can also be applied to study other histone posttranslational modifications and more generally to any discrete 3D signals.

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.

Microcystin-LR (MCY-LR), a widespread natural cyanobacterial toxin is a potent inhibitor of type 1 and 2A protein phosphatases (PP1, PP2A), inducing oxidative stress as well. Diquat (DQ) is a herbicide that inhibits electron transport to induce strong oxidative stress. Both compounds are affecting natural environments as well as plant and human health. We conducted a comparative analysis on their long-term (48 h) effects on reactive oxygen species (ROS) production, mitosis and histone H3 phosphorylation in root apical meristems of wild-type and PP2A-related mutants of Arabidopsis thaliana. MCY-LR did not increase ROS levels in any of the genotypes, but increased significantly the activity of ROS scavenging peroxidases (PODs) in Col0. DQ decreased ROS levels, but did not affect PODs. The paradoxical effect of DQ indicated the initiation of cell death processes. Mitosis was not affected by MCY-LR, but inhibited by DQ. Interestingly, MCY-LR as a phosphatase inhibitor did not affect histone H3 phosphorylation in mitotic cells, but DQ had an increasing effect. As we have shown previously, most of the above parameters changed differently at shorter (24 h) exposures: both compounds increased ROS levels in the phosphatase mutants, while POD activities were not affected; inhibition of mitosis or arrests in early mitosis, as well as increases of histone H3 phosphorylation levels occurred in the presence of both compounds. To conclude, Arabidopsis is able of recover partially and transiently from MCY-LR induced stress at long-term exposure, but this is not true for DQ. The environmental relevance of these findings is discussed.