Distinct Clades of Protein Phosphatase 2A Regulatory B’/B56 Subunits Engage in Different Physiological Processes

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B’ (B56), and B’’. Here, the Arabidopsis B’ group is reviewed and compared with other eukaryotes. Members of the B’α/B’β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B’ subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B’ subunits; B’α and B’β being of little importance, whereas others (B’γ, B’ζ, B’η, B’θ, B’κ) promote transition to flowering. Interestingly, the latter B’ subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.publishedVersio

Loss of LEUCINE CARBOXYL METHYLTRANSFERASE 1 interferes with metal homeostasis in Arabidopsis and enhances susceptibility to environmental stresses

The biochemical function of LEUCINE CARBOXYL METHYLTRANSFERASE 1 (LCMT1) is to transfer a methyl group from the methyl donor S-adenosylmethionine (SAM) to the catalytic subunits of PROTEIN PHOSPHATASE 2A (PP2Ac), PP4 and PP6. This post-translational modification by LCMT1 is found throughout eukaryotes from yeast to animals and plants, indicating that its function is essential. However, Arabidopsis with knocked out LCMT1 still grows and develops almost normally, at least under optimal growth conditions. We therefore proposed that the presence of LCMT1 would be important under non-optimal growth conditions and favoured plant survival during evolution. To shed light on the physiological functions of plant LCMT1, phenotypes of the lcmt1 mutant and wild type Arabidopsis were compared under various conditions including exposure to heavy metals, variable chelator concentrations, and increased temperature. The lcmt1 mutant was found to be more susceptible to these environmental changes than wild type and resulted in poor growth of seedlings and rosette stage plants. Element analysis of rosette stage plants mainly showed a difference between the lcmt1 mutant and wild type regarding concentrations of sodium and boron, two-fold up or halved, respectively. In both lcmt1 and wild type, lack of EDTA in the growth medium resulted in enhanced concentration of copper, manganese, zinc and sulphur, and especially lcmt1 growth was hampered by these conditions. The altered phenotype in response to stress, the element and mRNA transcript analysis substantiate that LCMT1 has an important role in metal homeostasis and show that functional LCMT1 is necessary to prevent damages from heat, heavy metals or lack of chelator.publishedVersio

Antagonistic regulation of flowering time through distinct regulatory subunits of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) consists of three types of subunits: a catalytic (C), a scaffolding (A), and a regulatory (B) subunit. In Arabidopsis thaliana and other organisms the regulatory B subunits are divided into at least three non-related groups, B55, B' and B″. Flowering time in plants mutated in B55 or B' genes were investigated in this work. The PP2A-b55α and PP2A-b55β (knockout) lines showed earlier flowering than WT, whereas a PP2A-b'γ (knockdown) line showed late flowering. Average advancements of flowering in PP2A-b55 mutants were 3.4 days in continuous light, 6.6 days in 12 h days, and 8.2 days in 8 h days. Average delays in the PP2A-b'γ mutant line were 7.1 days in 16 h days and 4.7 days in 8 h days. Expression of marker genes of genetically distinct flowering pathways (CO, FLC, MYB33, SPL3), and the floral integrator (FT, SOC1) were tested in WT, pp2a mutants, and two known flowering time mutants elf6 and edm2. The results are compatible with B55 acting at and/or downstream of the floral integrator, in a non-identified pathway. B' γ was involved in repression of FLC, the main flowering repressor gene. For B'γ the results are consistent with the subunit being a component in the major autonomous flowering pathway. In conclusion PP2A is both a positive and negative regulator of flowering time, depending on the type of regulatory subunit involved

Analysis of interactions between heterologously produced bHLH and MYB proteins that regulate anthocyanin biosynthesis: Quantitative interaction kinetics by Microscale Thermophoresis

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Expression levels of flowering control genes after complementation of the <i>pp2a-b’γ</i> mutant.

<p>Shoots were harvested ten days after germination, 12 h into the 16 h photoperiod. Gene expression was tested in WT, and the mutants, <i>pp2a-b’γ, pp2a-b’γ-complemented</i>, and e<i>lf6</i> (control). Genes tested were: <b>a</b>) <i>FLC</i>, <b>b</b>) <i>SOC1</i>, and <b>c</b>) <i>FT</i>. The <i>pp2a-b’γ</i> mutant showed normal WT expression levels when complemented with the <i>35S-PP2A-B’γ</i> gene construct. Data presented are means of three independent experiments for <i>FLC</i> expression and two for <i>SOC1</i> and <i>FT</i>. Each sample contained 50 plants and was assayed in triplicate. Vertical bars indicate the standard error of the mean.</p

Visible phenotypes of representative <i>pp2a</i> mutants.

<p><b>a</b>) Representative plants of WT and early flowering mutant lines <i>pp2a-bα</i> (SALK_09504) and <i>pp2a-bβ</i> (SALK_062614). <b>b</b>) Mutant line <i>pp2a-b’γ</i> complemented with 35S::PP2A-B’γ showing flowering time as WT, late flowering line <i>b’γ</i> (SALK_039172), and WT plants. Plants were grown in 16 h days.</p

Expression levels of genes important in different flowering time controlling pathways and the floral integrator.

<p>Shoots were harvested ten days after germination, 12 h into the 16 h photoperiod. Gene expression was tested in WT and the mutants <i>pp2a-bα SALK_09504</i>, <i>pp2a-bβ SALK_062614</i>, <i>pp2a-b’γ</i>, e<i>lf6</i> (early flowering control) and <i>edm2</i> (<i>late flowering control</i>). Genes tested were: <b>a</b>) <i>FLC</i>, <b>b</b>) <i>CO</i>, <b>c</b>) <i>MYB33</i>, <b>d</b>) <i>SOC1</i>, <b>e</b>) <i>FT</i> and <b>f</b>) <i>SPL3</i>. Expression of established flowering pathway genes are modulated in <i>pp2a-b’γ</i> consistent with this mutant being late flowering, whereas <i>pp2a-b55</i> mutants show only minor changes in transcript levels and may act on flowering time through an unknown pathway. Data presented are means of three (except for <i>SPL3</i>, which had two) independent experiments of samples each containing 50 plants and assayed in triplicate. Vertical bars indicate the standard error of the mean.</p

Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation1[W][OA]

Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55β. Interestingly, the homozygous double mutant (b55α × b55β) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bβ and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation

Protein phosphatase 2A regulatory subunits are starting to reveal their functions in plant metabolism and development

Canonical protein phosphatase 2A (PP2A) consists of a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to confer substrate specificity and cellular localization to the PP2A complex, and are generally divided into three non-related families in plants, i.e., B55, B′ and B″. The two Arabidopsis B55 subunits (α and β) interact with nitrate reductase (NR) in the bimolecular fluorescence complementation assay in planta, and are necessary for rapid activation of NR. Interestingly, knockout of all four B55 alleles is probably lethal, because a homozygous double knockout (pp2a-b55αβ) could not be found. The B55 subunits, therefore, appear to have essential functions that cannot be replaced by other regulatory B subunits. A double mutant (pp2a-b′αβ) of two close B′ homologs show severely impaired fertility, pointing to the essential role also of B′ subunits in plant development